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for mutagenic activity. 30, 132, 167, 168, 171 Wang et al. 177 collected air samples from a res idential area at an intersection of two heavily trafficked crossroads in the Buffalo, New York, area. Extraction of the particulate fraction with acetone resulted in a preparation highly mutagenic in Salmonella strains TA 98, TA 100, and TA 1537. These investigators also obtained a positive direct mutagenic response with automobile-exhaust samples from a spark-ignition internal-combus Lion engine (with leaded gas as the fuel). The mutagenic ingredients appeared to originate in motor oil during the combustion process and were not due to lead. Similar results have been obtained by Pitts et al.132 with atmospheric particulate extracts from the Los Angeles basin, by Teranishi et al. , 168 by Tokiwa et al. 171 with extracts from several Japanese cities, and by Talcott and Weil67 and Commoner et al.30 with extracts from other American cities. Unfortunately, the quantitation of some of these studies may be open to question because of filter artifacts. The disposition of the filter apparatus in relation to sunlight, temperature, etc., is important, because these factors may facilitate chemical reactions involving PAHs and may result in artifactual formation of mutagens. This aspect is discussed in Chapter 3. Soot makes up 2-15: of the mass of fine particles that are present in urban atmospheres.105 A number of studies have been conducted to establish its mutagenic potential. Kadin et al .83 have experimen- tally generated soot from ingredients with varied sulfur composition --i.e., from pyridine, decalin, and o-xylene or from th~iophene, decalin, and o-xylene--and have compared its mutagenicity with that of soot obtained from burned kerosene. Dichloromethane extracts of all the soots were mutagenic in a bacterial assay in which a forward muta- tion of 8-azaguanine resistance was measured. The soots generated from the sulfur-containing and nitrogen-containing ingredients, as well as soots from kerosene or furnace black, exhibited 10-17: of the mutagenic activity of authentic BaP (on a weight basis ~ . Emission from spark-ignition combustion and diesel engines has been tested for mutagenic activity in the Salmonella system.27~79~83~10l It is known that particulate emission from light-duty diesel engines is considerably greater than that from light-duty catalyst-equi~ped spark-ignition engines--i.e., 0.2-1.0 vs. 0.006-0.02 g/mi.l4 Table 4-8 presents data of Claxton 8 relative to comparative mutagenic activity of emission of diesel and spark-ignition engines, of cigarette-smoke condensate, of coke-oven emission, of roofing-tar emission, and of BaP (positive control). The results are reported in terms of revertants/100 fig of soluble dichloromethane organic compounds; the soluble organic components represent approximately 25% of the total mass of the particles. As is evident from the table, cigarette-smoke condensate, roofing tar, and BaP required metabolic activation by an S-9 fraction, whereas diesel-engine exhaust was directly mutagenic. The other kinds of emission were both directly and indirectly mutagenic. The diesel exhaust exhibited a wide range of mutagenic activity, although the high value is probably peculiar to the 4-11
particular engine that generated the emission. far greater than that of emission. The activity of BaP is Naman and Clarkll9 have determined the quantity of particles emitted and the mutagenic activity of extracts of the exhausts of several spark-ignition engines that burned gasoline, a 901/10% ethanol blend, or commercial gasohol. The results are presented in Table 4-9. Although the number of rever ten ts per mile differed for each of the four-cylinder engines, the addition of ethanol clearly reduced the direct mutagenic capacity. Several investigators have determined the mutagenic activity of respirable coal fly ash,25~69 which does have mutagenic activity in the Salmonella/microsome assay. Virtually all the emissions yield a mutagenic response in this test system. Extracts from the various kinds of emission contain a large number of PAHs, among which is BaP.48~89~176 Extracts of diesel particles have been separated on Sephadex LH-20 into six fractions;61 the contribution of each to the total mass of the diesel extract obtained from a low-sulfur and high-sulfur fuel is shown in Table 4-10. Furthermore, each of these exhausts was obtained before or after passage through an oxidative catalyst. Fraction 1 contained most of the mass of the extracts from both fuel exhausts. However, fractions 3 and 4 contained most of the mutagenic activity. Fraction 3 from the low-sulfur exhaust contained the bulk of the PAHs, including phenanthrene, methylphenanthrenes, fluoranthene, pyrene, methylpyrenes, benzo[ghi]fluoranthene, benzanthracene (BA), chrysene (or benzo[c]phenanthrene), methyl-BAs, and BeP (or perylene).61 With the high-sulfur fuel, one found, in addition, the methylbenzothiophenes. It is of interest that the low-sulfur fuel gave an exhaust whose mutagenic activity was increased after passage through a catalyst. The reverse was true for the high-sulfur fuel. Furthermore, fraction 4 from the high-sulfur fuel, before oxidative catalysis, proved the most mutagenic. The major identified components of emission have been tested for mutagenic activity with the Salmonella forward-mutation assay of Thilly and co-workers.83~10l In this assay, mutants that are resistant to the purine analogue 8-azaguanine are scored. Of the components present in kerosene-soot extract, cyclopenta[cd]pyrene proved the most mutagenic; it was also present in the highest concentration (see Table 4-11~. C~cloyentatcd~pyrene is a known component of all soots, 53' 74' 75 of cigarette smoke,l59 of automobile exhaust,57 and of coal fly ash.25 The sum of the mutagenicities of the identified individual PAHs was slightly greater than that of the kerosene-soot extract itself. The total mutagenic activity of the kerosene-soot extract could almost be reproduced by that of the cyc lopenta ~ cd ~ pyrene . The investigators compared the mutagenic efficacy of additional PAHs with and without an S-9 preparation, using induced cells from the liver; the results are in Table 4-12. Methylation of several of the 4-12
inactive PAHs, such as anthracene and phenanthrene, resulted in the acquisition of mutagenicity. Preliminary evidence has led the Thilly group to suspect the presence of alkyl-substituted anthracene and phenanthry~ in diesel-soot fractions that were mutagenic in bacteria. In this series, the most active compound was perylene, which was followed by cyclopenta [cd] pyrene. The mutagenicity of cyclopenta ~cd] pyrene in the Salmonglla/microsome assay has been confirmed by Eisenstadt and Gold;4 metabolic activation by the S-9 fraction was required before this mutagenic property was elicited. Ni treated PARE As mentioned previously, emission from either diesel or spark-ignition engines exhibits considerable direct-acting mutagenic activity in the Salmonella/microsome assay, whereas cigarette-smoke condensates, roofing-tar extracts, and BaP do not. This has led several investigators to study engine exhaust for the presence of direct-acting PAR derivatives that might have been produced by gaseous exhaust components--~.g., nitrogen oxides--or by atmospheric oxidative reactions involving ozone. Various nitropyrenes and other analogues have been assayed for mutagenic activity in the bacterial system 52,96,111,112,124,130 These substances exhibit potent activity in the Salmonella mutagenesis assay. Indeed, it has been estimated by Gorse (personal communication) that the concentration of nitropyrene alone in diesel particulate extracts could account for 13-24: of the total direct mutagenic activity with-TA 98. Tokiwa et al.170 have assayed the mutagenicity of the nitrophenanthrenes, 1-nitropyrene, 3-nitrofluoranthene, and 6-nitrochrysene. Each of the parent PAHs was inactive as a direct mutagen, but 6-nitrochrysene was slightly active, nitrophenanthrene was active, and 1-nitropyrene was most active against TA 98 and TA 100. S-9 was not required for this demonstration of mutagenic activity. Pitts et al .132 reported the direct mutagenic activity of 1-, 3-, and 6-nttrobenzo~aipyrene in the Salmonella/microsome assay. Perylene, another exhaust constituent that is converted to 3-nitroperylene, demonstrated mutagenesis.132 In a similar fashion, nitrated derivatives of anthracene, fluoranthene, benz~aJanthracene, benzo~kifluoranthene, and benzotghi~perylene--all of which are present in diesel exhaus t--exhibited potent mutagenic activity in the Salmonella assay.16 The nitropyrenes have been reported as contaminants of xerographic copiers and toners which may therefore contribute to the problem of mutagenicity. 103 ~ 143 Rosenkranz et al . 143 have demonstrated the presence of such a mutagenic activity with various Salmonella test s trains; they have traced this property to nitropyrenes that were present as impurities in carbon black. In addition to mononitrated components, they were able to identify the 1,3-, 1,6-, and 1,8-dinitropyrenes, 1,3,6-trinitropyrene, and 1,3,6,8-tetranitropyrene as contaminants. All these derivatives demonstrated direct mutagenic activity (see Table 4-13) with both nitroreductase-positive and -negative variants of Salmonella. The mutagenic property of 4-13
1-nitropyrene and 1,3-dinitropyrene depended heavily on the endogenous bacterial nitroreductase activity. Insertion of two nitro groups in the pyrene moiety increased mutagenic activity by a factor of approxi- mately 100, although information is ins uf ficient to extrapolate to other PAHs. The most potent of these derivatives was 1,8-dinitro- pyrene. It was striking that, of the three dinitro derivatives, two ac t ed independent ly 0 f endogenous n i troreduc base; equa 1 numbers 0 f revertants per nanomole are observed in both TA 98 and TA 98 NR. A similar situation occurred with the trinitropyrenes and tetranitro- pyrene. The mutagenic activity of 1,8-dinitropyrene is the highest ever recorded in the 1 iterature, 112 The presence of the 1, 6- and 1,8-dinitropyrenes as predominant mutagenic components in diesel- particle extract has been confirmed by Pederson and Siak,124 who estimated that 15-20: of the total mutagenic activity of the extract may be contributed by these dinitropyrenes (in addition to as much as 24t contributed by 1-nitropyrene). Sulfur-Containin~,PAHs The presence of sulfur-containing heterocyclic PAHs has been reported in various combustion products, particularly from high-sulfur petroleum products (see Chapter 1). In many heterocyclic structures, one aromatic ring has been replaced by thiophene.86 It is anti- cipated that the increase in the use of coal, particularly with high sulfur content, will result in substantial environmental pollution with these ingredients . Thus, it is imperative to have a bet ter understand- ing of the biologic effects of these sulfur-containing heterocyclic PAHs. The mutagenic ity of several s ul fur-containing PAHs has been determined in the Salmonella/microsome assay by Karcher et al.,87 and the results are presented in Table 4-14 . Of the isomers listed, benzo[2,3Jphenanthro[4,5-bcd] thiophene was the most potent; its ring configuration corresponds to that of BaP, although it is more mutagenic than the la t ter . ANIMAL-CELL MUTAG~.N~.~ T ~ A number of animal-cell model systems have been used to ascertain the mutagenic effects of combustion-engine emission, as well as other exhaul7' 1 ~ ese have been reviewed in previous monographs on PAHs. ~ Many of the tests depend on the selection of variants on the basis of resistance to 8-azaguanine, 6-thioguanine, ouabain, or deoxythymidine analogues. Comparative data on the development of 6-thioguanine resistance in Chin22e hamster ovary (CHO) cells have been reported by Casto et al., who used extracts of diesel-exhaust particles and coke-oven emission (see Table 4-15~. All extracts yielded the same number of 4-14
mutant cells, which was comparable with that of the positive control, methyl methanesulfonate, a known direct-acting methylating agent. Curren et al.34 have tested the production of ouabain-resistant BALB/c 3T3 cells when the latter were exposed to a variety of agents (see Table 4-16~. The spark-ignition-engine extract was considerably more mutagenic in this assay than the diesel extracts. A roofing-tar pot sample and coke-oven emission also exhibited greater mutagenic efficacy. The presence of an activating system did not significantly affect mutagenicity. The extract from the gasoline-engine exhaust appeared more mutagenic than the extracts from various diesel engines, and coke-oven pot samples were even more active. Mutability at several different genetic loci by PAHs has been determined by Huberman and Sachs78 (Table 4-17~. Good mutagenic activity with respect to the HGPRT locus was manifested by dibenz~acianthracene, dibenz~ah~anthracene, 7-methylbenzanthracene, BaP, 7,12-DMBA, and 3-MC. The last four compounds named were also mutagenic with respect to ouabain resistance. At both loci, 7,12-DMBA was most active. As indicated previously, diesel exhaust demonstrates considerable direct mutagenic activity in the Salmonella/microsome assay. The nitro-PAHs have been considered as likely candidates for this activity. Thilly and colleaguesl°l have been unable to demonstrate any direct mutagenic activity with human lymphoblasts as the target cells, although, in the presence of an activating system, a consider- able amount of 6-thioguanine resistance and trifluorothymidine resistance resulted after addition of diesel extract to the culture media. These experiments suggest that the nitrated PAHs, if present in the diese1 extracts, are rapidly inactivated by the lymphoblasts or require for activation a nitroreductase (or other enzyme) that is absent from these cells. Indeed, application of the term "direct- acting mutagen" to the nitrated PAHs is not entirely correct. It is postulated that these analogues undergo a reduction, catalyzed by a nitroreductase, to an amino derivative that may be further transformed into reactive hydroxylamino PAHs (see Chapter 3~. The latter would easily form electrophilic substances that could interact with DNA in causing a mutation. What is needed is additional experimentation on the mechanism of action of the nitrated PAHs in both bacterial and mammalian-cell systems. Sister chromatic exchange has been used to assess genotoxic activity of various kinds of emission. Unfortunately, SCE appears to be more predictive of point mutation than of frameshift mutation,l9 whereas most of the PAHs produce the latter damage. The experiments of Mitchell et al ., 116 which used CHO cells, indicated that all the emission extracts were inferior to BaP in inducing SCE. Of the emis- sions, coke-oven extracts proved the most active, and the heavy-duty Caterpillar dresel-engine exhaust was the least potent. Intermediate in activity were cigarette-smoke condensate, roofing-tar emission, Mustang gasoline-engine emission, and other diesel-engine emission. None of these required metabolic activation for SCE activity. 4-15
The induction of SCE has been performed in viva with Chinese hamsters that were given various PAHs intraperitoneally.1 After two injections, the bone marrow was aspirated, and the SCEs per metaphase cell were determined. Although the positive control, BaP, did produce SCE, there was little correlation between the quantitative aspects and the carcinogenic potential of the PAHs. No comparable experiments were performed with the various kinds of emission. The experiments of Schonwald et al.150 also showed a lack of correlation between carcinogenicity and ~E. These investigators determined SCE induced by BaP with human lymphocytes obtained from normal persons and lung-cancer patients; no difference was observed. Guerrero et al.58a intratracheally exposed Syrian hamsters to 200 ng of BaP over a 10-wk period, examined in vitro cultures of lung tissue for sister chromatic exchange (SCE), and concluded from the results that BaP was metabolically activated by lung cells in viva. In other studies, diesel exhaust particles (DEP) in doses of 0-20 mg per hamster were administered over a 24-h period; although the study was limited in scope, the results demonstrated that DEP can induce genotoxic damage. CARC INOGENE SIS SKIN Kotin and colleagues89~91 first reported the presence of carcino- genic substances in the exhaust of gasoline and diesel engines. Benzene extracts of particles from these sources produced both papillomas and carcinomas when applied to the skin of mice. These studies were extended by Wynder and Hoffmann,l82 who compared the carcinogenicity of cigarette tar with that of organic extracts of gasoline-engine exhaust particles. The latter, obtained from a 1958 gasoline engine without a catalytic converter, proved twice as active (on a weight basis) as cigarette tar. Many studies have since been conducted with skin as the target tissue; only a few are described here. Automobile-exhaust condensate has been partitioned into a number of fractions by Pott et al.,135 with the PAHs predominantly found in fraction IV, the nitromethane phase. Each of these fractions was testedl5 for ability to produce papillomas and carcinomas in life- long mouse skin-painting experiments in which combined initiator and promoter activity was measured. BaP, the positive control, at 1.92-7.68 ~g/treatment caused tumor formation in 15-60% of the mice. The exhaust condensate at 0.53-4.7 mg/treatment, equivalent to BaP at 0.15-1.35 ~g/treatment, produced tumors in 1-72% of the mice, and the tumors arose after a shorter latent period. The major tumor-producing activity was noted in fraction IV, which contained the PAHs. In this fraction, however, BaP is responsible for only 91 of the carcino- genicity of automobile-exhaust condensate (AEC). 4 Agents other than BaP, acting either alone or synergistically with AEC, are responsible for the major carcinogenicity of AEC and probably of diesel exhaust. The tumor-producing effects of AEC in the carcinogen mouse model have been contrasted with those of 15 PAHs that occur as major 4-16
components of AEC.~14 These components and their relative concentrations in a simulated AEC mixture are shown in Table 4-18. AEC, diesel-exhaust condensate (DEC), BaP (the positive control), and the mixture of PAHs were tested for their comparative potency (see Table 4-19~. The data indicate the greater potency of AEC than of DEC. If the relative potency of AEC were accepted as 1, the corresponding values for DEC, BaP, and the PAR mixture would be 0.02, 187, and 68, respectively. The proportions of the carcinogenic potency of AEC and DEC attributable to the selected PAHs can be calculated. BaP would account for only 9.6% of this potency in AEC, and the selected PAHs, only 41%. In DEC, the contribution of BaP is approximately 161. These results indicate that compounds other than the selected PAHs contribute to the carcinogenic potency of AEC or DEC. Slaga and associatesl58 used a mouse that had been bred for quickness of response in the initiation-promotion skin-carcinogenesis model--the SENCAR mouse--to study comparative biologic potency of various kinds of emission and PAHs (see Table 4-20~. The exhausts were relatively ineffective, in comparison with purified BaP, in causing papilloma formation. Indeed, 10 mg each of emission from roofing tar, coke ovens, and the Nissan diesel engine was equivalent in response to 50, 60, and 80 g of BaP, respectively. In no case did 10 mg of emission extract contain that much BaP. The activity of anthracene, pyrene, dibenz~ah~anthracene, dibenz~acianthracene, benz~aJanthracene, 2-hydroxybenzota~pyrene, and BaP as complete carcinogens and as tumor initiators was compared in this mouse strain. 158 Their relative potencies were 0, 0, 20, 0, 5, 30, and 30, respectively, compared with 7,12-DMBA, set at a potency of 100. Schmahl and colleagues extended these studies by determining whether groups of nonactive PAHs would interact with the carcinogens in a synergistic or inhibitory manner.149 The proportions of the various compounds were chosen on the basis of their relative concentrations in automobile exhaust. The groups of carcinogens and noncarcinogens are shown in Table 4-21, and the percent tumor formation after lifetime application is shown in Table 4-22. Mixtures of the four carcinogens were more effective than a comparable dose of BaP alone. Of greater importance, no evidence of synergism or inhibition could be found when mixtures of carcinogens and noncarcinogens were applied. The application of multiple PAHs to mouse skin has often resulted in data that were confusing, with regard to carcinogenesis. Thus, in opposition to the above discussion, Steinerl60 reported that the combination of two weak carcinogens, benz~ajanthracene and chrysene, resulted in a synergistic-tumorigenic response; benz~aJanthracene and dibenz~ahianthracene yielded fewer tumors than expected; and dibenz~ahianthracene and 3-MC yielded the sum of individual tumorigenic potentials. Falk and co-workers47 reported much lower tumor production after the simultaneous administration of BaP and3severa noncarcinogenic hydrocarbons. Van Duuren and Goldschmidt17 noted that repeated application of the weak carcinogen BeP and the noncarcinogen pyrene to mouse skin with BY resulted in a cocarcinogenic effect. DiGiovanni et al. 1 found that mouse-skin carcinogenesis induced by 7,12-DMBA-~is inhibited when BeP, pyrene, or fluoranthene was applied 5 min before the initiator. The apparent paradox was explained by the later studies of DiGiovanni and 4-17
Slaga.40 They used either 7,12-DMBA, BaP, or 3-MC as an initiator and tetradecanoyl phorbol acetate (TPA) as the promoter. BeP or dibenz~ac~anthracene was applied 5 min before the initiator in all cases. With 7,12-DMBA as the initiator, BeP and dibenz~acianthracene each reduced tumorigenes is by more than 80%. However, with BaP as . . . initiator, dibenz~ac~anthracene exerted no effect and BeP stimulated tumor formation by 30%. If dibenz~ac~anthracene was applied 12, 24, or 36 h before BaP, a reduction in tumorigenesis was observed. With 3-MC as initiator, dibenz~acianthracene inhibited tumor formation, whereas BeP was without effect. BeP apparently exerts its effect on the 7,12-DMBA-initiated system by profoundly inhibiting the ring hydroxylation of this initiator and reducing the covalent binding to DNA. Thus, the order of application of the multiple noncarcinogenic with carcinogenic PAHs can have serious effects on carcinogenesis. Finally, with regard to mouse-skin tumorigenesis, cyclopentatcd]- pyrene, a major component of soot that can transform mouse fibroblasts oncogenically,122 was tested for tumor-initiating activity on mouse skin by Wood et al. 181 Although tumorigenic, cyclopentatcdipyrene was weaker than BeP. TISSUES OTHER THAN SKIN . . . PAHs and exhaust condensates have been administered to experimental animals in ways other than topically. The subcutaneous injection of AEC and fractions thereof into mice produced sarcomatous lesions;135 administration of 20-60 mg yielded tumors in up to at of mice, and administration of 10 or 90 fig of BaP yielded tumors in 17t or 75t of the animals, respectively. Simultaneous administration of 20 mg of AEC with 90 fig of BaP yielded lower tumorigenesis. The most active fraction from AEC was the nitromethane phase, which contained the various PAHs. Sellakumar and Shubikl5l studied benz~aJanthracene, benzo~b]- fluoranthene, dibenz~ah~anthracene, dibenzotai~pyrene, and pyrene. They mixed the PAHs with a hematite dust (at 1:1), suspended the mixture in 0.9% saline, and instilled it intratracheally at weekly intervals into Syrian golden hamsters. Most of the PAHs were not carcinogenic in this limited series , but dibenzotai~pyrene produced a high incidence of carcinomas. With multiple doses that totaled 8 mg of this subs Lance, 47: of the hams ters had respiratory tract tumors (squamous cell carcinomas); with 12 ma, 89% of the animals were affected. This degree of carcinogenicity is greater than that of BaP. Reznik-Schuller and Mohrl37 have compared the carcinogenicity of AEC with that of several major PAR constituents in the Syrian golden hamster intratracheal model. The hamsters were given AEC at 2.5 or 5 mg/animal every 2 wk intratracheally, corresponding to a total administration of 42.5-75 or 75-150 mg of AEC. The total was equivalent to 11.56-25.5 or 25.5-51 fig of BaP. In all animals, multiple pulmonary adenomas were observed. This strikingly high incidence of neoplasia could not be explained by the BaP content of the AEC, It is of interest, however, that no carcinomas were observed. 4-18
As indicated earlier and as is discussed more fully in Chapter 6, ingestion of PAHs, whose presence may be attributed to vehicular exhaust, appears to be a ma jor route of entry in animal systems. Yet, the literature pertinent to this form of administration of exhaust particles, their major PAHs, and mixtures thereof is very limited. Neal and Rigdonl2l, 40 have examined the effects of oral administra- tion of BaP on tumor formation in mice. No gastric tumors developed in any of the 289 mice that were fed a control ration; the incidence of tumors in the BaP-fed mice depended on concentration in the food and on the number of days of feeding.l2l These investigatorsl40 also established that the incidences of pulmonary adenomas, gastric tumors, and leukemia in BaP-fed mice were genetically determined. No relation- ship, however, was observed between the relative incidences of these two types of neoplasms within a given mouse. Studies of these types would be useful, with regard to other PAHs and their mixtures. The interpretation of these studies is colored by the failure to house the mice in metabolic chambers, which would eliminate the contribution of coprophagy. Another series of studies took advantage of the susceptibility of the A strain mouse to pulmonary adenoma formations particularly after the intravenous administration of selected PAHs.1 3 Shimkin and Stonerl53 were able to calculate the amount of each agent that had to be injected for the induction of one pulmonary adenoma in this strain of mouse. The compounds tested were 3-MC, dibenz~ah~anthracene, 7H-dibenzotcgicarbazoyl, BaP, dibenz~aj~aceanthrylene, and dibenz~ah]- acridine. The respective values were 0.9, 1.O, 6.0~, 9.5, 14, and 18 mol/kg of body weight for one adenoma. Benz~aJanthracene was essen- tially inactive. The objection to the use of the A strain mouse for these types of studies rests on its extraordinary sensitivity to pulmonary adenoma formation. In fact, if the A strain mouse is allowed to survive long enough, almost all the untreated animals will develop these tumors--they are already "initiated. " ALKYLATED PAHs, MUTAGENESIS, AND CARCINOGENESIS . Because of the presence of alkylated PAHs in cigarette smoke and various coal-derived liquids and tars,56362371373 their biologic effects are of paramount interest. Perhaps the most thoroughly studied of the alkylated PAHs are the methylbenz~aianthracenes, methylchrysenes, methylanthracenes, and methylphenanthrenes. Some of the earliest studies, in which the effects of a methyl group on the carcinogenicity of benz~ajanthracene (BA) were investigated, were conducted by Dunning and Curtiss and by Huggins's laboratory.8 These investigators monitored sarcoma incidence in rats to which-the various PAHs had been administered subcutaneously. Their results, which were in remarkable agreement, indicated that the insertion of a methyl group at position 6 or 7 of BA increased tumorigenicity to the extent that 70-100t of the rats were affected. 4-19
However, 8- or 12-methyl-BA resulted in tumor formation in only 50-69% of the rats, and 1-, 2-, 3-, 4-, 5-, 9-, 10-, or 11-methyl-BA proved noncarclnogenlc. The nature of the alkyl group was an important consideration: substitution of an ethyl group at position 7 or 12 of BA greatly diminished tumor incidence, compared with that of the methyl congeners.123 Pataki and Huggins 123 have also studied the structure-activity relationship in the BA series when two methyl groups were inserted. A marked increase in tumorigenicity--shown by sarcoma formation--was observed with 6,7-dimethylbenz~aJanthracene (DMBA) and 6,8-, 6,12-, 7,8-, 7,12-, and 8,12-DMBA. But, 1,12-, 3,9-, and 9,10- OMBA were essentially nontumorigenic. Of the trimethylated BA derivatives, 6,7,8-, 6,7,12-, and 7,8,12-trimethylbenz~aJanthracenes were all very tumorigenic. The mutagenicity and tumor-initiating activity of methylated fluorenes, phenanthrenes, anthracenes, and benzofluorenes were studied by LaVoie et al.99~100 Only the 9-methylfluorene was more mutagenic in the Salmonella/microsome assay than the parent compounds; the 1-, 2-, 3-, and 4-methylfluorenes were as poor mutagens as fluorene itself. In the clime thyl series, 1,9-dimethylfluorene was a potent mutagen in Salmonella TA 100, and the 2,3- and 9,9-dimethylfluorenes were relatively ineffective. Benzo~a~fluorene, benzotb~fluorene, and benzo~cifluorene were poor mutagens in the organism, but the 11-methyl derivatives of the first two and the 7-methyl derivative of the latter were more effective. Of this series of methyl derivatives, 11-methylbenzo~bifluorene was the best mutagen. In the phenanthrene series, 99 only the 1- and 9-methyl analogues exhibited greater mutagenicity than phenanthrene itself. Equal mutagenic activity was manifested by phenanthrene, the 2-, 3-, and 4-methyl analogues, and the 3, 6- and 2, 7-dimethyl analogues . The poor mutagenic activity of anthracene was not altered by substitution of a methyl group in position 1, 2, or 9. Tumor-initiating activity of several of these alkylated PAHs was determined with the mouse-skin two-stage carcinogenesis model.l°° In a series of fluorene, 9-methylfluorene, 1,9-dimethylfluorene, benzota~fluorene, benzo~b~fluorene, benzotcifluorene, 11-methylbenzo~a~fluorene, 11-methylbenzotbifluorene, and 7-methylbenzotcifluorene, only 11-methylbenzotb~fluorene resulted in a marked increase in tumorigenicity. All other compounds exhibited rather weak initiator activity. The methylchrysenes are known respiratory pollutants that occur in substantial amounts in cigarette smoke--approximately 18 ng/cigarette.62 Although chrysene itself is generally inactive, several of the meghylated species are carcinogenic. In early studies, Gough and Shoppee 5 and Dunlap and Warren44 showed that the 1-, 4-, 4-20
and 6-methylchrysenes demonstrated only weak tumorigenicity. The 1,11-dimeth~1 derivative, however, was moderately active as a skin carcinogen, 2 although less so than 3-MC. Hecht and colleagues63 studied a series of methylated chrysenes as both complete carcinogens and initiators. As a complete carcinogen, 5-methylchrysene was far superior to chrysene and the other monomethylated derivatives; it was almost equivalent in carcinogenic potency to BaP. 2-Methylchrysene exhibited about 50% of the carcino- genicity of the 5-methyl analogue . As an initiator, 5-methylchrysene was also the most potent of the methylated derivatives, yielding tumors in 50% of the mice by 14 wk. Next in potency was 3-methylchrysene. The 1-, 4-, and 6-methylchrysenes were all much less effective as tumor initiators. These investigators63 considered whether 5-methyl- chrysene, rather than BaP, would be a major contributor to the carcino- genicity of tobacco smoke; but, in view of its small concentration in tobacco smoke, compared with that of BaP (0.6 ng/cigarette vs. 30 ng/cigarette), it is unlikely that this is so. A series of methylated BaPs were tested for tumor-initiating activity with the mouse-skin carcinogenesis model.157 Several of the methylated derivatives exhibited greater initiating activity than the parent compound, namely, the 1-, 3-, and 11-methyl analogues. Several were completely ineffective in this regard: the 7-, 8-, 9-, -and 10-methyl analogues. The 4-methyl derivat ive was about equal to BaP in initiating potency. From these examples, it is apparent that some methylated PAHs are strong carcinogens and therefore should be reckoned with as environ- mental contaminants. TOBACC O- SMOKE CARC INOGENE S I S . Although the topic has been discussed extensively, several of the potent carcinogenic PAHs that are present in tobacco smoke should be mentioned here . Approximately half of tobacco smoke consists of particulate consti- tuents in which over 2,000 compounds are represented. The carcino- genicity of cigarette smoke was demonstrated ~hrough skin application to the backs of mice and the ears of rabbits7 and has been confirmed repeatedly in a number of laboratories. Unfortunately, inhalation experiments have not led to as clear-cut a conclusion. When Syrian hamsters were exposed to diluted smoke (smoke-to-air ratio, 1:7) for 10 min twice a day for 18 mo, precancerous lesions were observed in 30%, esophageal tumors in 5t, and laryngeal carcinomas in 10% of the animals; no bronchial or tracheal cancer was seen.72 Cigarette-smoke condensates have been partitioned into a number of fractions, of which the most carcinogenic is the "neutral fraction," representing 57: of the mass of the condensate.72 Although the
weakly acidic fraction (approximately 2: of the condensate mass) con- tained little carcinogenicity itself, 80% of the tumorigenic property of the total condensate could be reproduced in conjunction with the neutral fraction. The neutral fraction was further fractionated by silica-gel chromatography and partitioning between n-hexane and nitro- methane into a preparation that contained 0.6% of the total mass, but much of the carcinogenicity. The active nitromethane preparation was further fractionated into two components, each of which contained PAHs. The relative tumor-initiating activity and concentration of several of these ingredients are shown in Table 4-23. BaP, dibenz~ah]- anthracene, benzotbifluoranthene, benzo~j~fluoranthene, and dibenz~a]- acridine--all present in substantial amounts in cigarette-smoke conden- sate--are potent carcinogens. Cocarcinogenicity was demonstrated by applying cigarette-smoke "tar" to the backs of mice in combination with a mixture of 17 major PAHs found in smoke. The concentration of the 17 PAHs was such as not to be tumorigenic. However, the combination of tar and PAHs resulted in tumor formation in 55% of the mice at 13 wk. whereas tar alone yielded tumors in only 18% of the mice. It should be mentioned that various neutral fractions obtained from the cigarette-smoke condensate significantly increased the tumorigenicity of BaP applied topically to mice . SUMMARY OF ANDMAL-CELL MUTAGENESIS AND CA-RCINOGENESIS DATA Although extracts of automobile emission demonstrate mutagenicity in both bacterial and animal-cell systems, their carcinogenicity is not great. Furthermore, attempts to reproduce their pharmacologic activity by assembling mixtures of the major PAR constituents have not been successful. The activity of the major PAHs as carcinogens or mutagens is depicted in summary fashion in Table 4-24. The most potent derivatives in eliciting a mutagenic response in the Salmonella/ microsome assay are the nitropyrenes. These have not been tested for carcinogenicity. However, a number of PAHs, such as BaP and some benzofluoranthenes (Table 4-24), are potent carcinogens. There is moderate agreement between the mutagenicity and carcinogenicity of the individual PAHs, although some exceptions are apparent, e.g., fluoranthene. A better predictor of carcinogenicity would consist of a battery of four tests--assays for mutation, chromosomal aberration, primary DNA damage, and morphologic transformation. 4-22
TABLE 4-l Categories of Short-Tenm Testsa Category Tests in bacteria and phage Tests in eukaryotic microorganisms Mammalian-cell mutagenesis tests In vitro transformation tests No. Methods Identified 13 19 21 18 Tests of DNA repair and other effects 14 In viva tests in mammals Tests in insects Mammalian cytogenetics tests 14 4 13 aData from Hollstein et al.74 Many assays detect the same genetic event, but are considered separate systems because of other differences, such as in target organism or cell line. Decisions to regard methods sufficiently distinct to be considered separately are arbitrary. 4-23
TABLE 4-2 Representative Short-Term Screening Systems Bacteria tests: Cytogenetics tests Salmonella/microsome test Poly A test (E. coli) Yeast tests: Mitotic recombination or gene conversion (Saccharomyces cerevisiae) . . Mammalian mutagenesis tests: Mouse lymphoma TK +/ CHO/HGPRT Chinese hamster V79 BALB 3T3 OuaR Insect test: Drosophila sex-linked reces- sive-lethal test In viva tests in mammals: Sperm-abnormality test Dominant-lethal test Mouse specific-locus spot test 4-24 Sister chromatic exchange · ~ In VlVO Sister chromatic exchange in vitro Chromosomal aberrations in viva Chromosomal aberrations in vitro Micronucleus test Tests of DNA effects and other effects: Unscheduled DNA synthes is (UDS) in human fibroblasts UDS in hepatocyte primary- culture/DNA-repair tests UDS in other target cells In vitro transformation tests: Baby hamster kidney cells BALB 3T3 and other cells Hamster embryo cells Enhancement of viral trans- formation
TABLE 4-3 Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) Short-Term Battery For detecting gene mutations, three of the following: Bacterial mutagenesis assay, with and without activation Eukaryotic microorganisms, with and without activation Insects (sex-linked recessive-lethal test) Mammalian somatic cells in culture, with and without metabolic activation Mouse specific-locus test For detecting chromosomal aberrations, three of the following In vivo cytogenetics tests in mammals Insect tests for heritable chromosomal effects Dominant lethal effects in rodents Heritable-translocation tests in rodents For detecting primary UNA damage, two of the following: DNA repair in bacteria, with and without activation Unscheduled DNA synthesis (repair test) in mammalian cells, with and without act iva Lion Mitotic recombination and/or gene conversion in yeast cells, with and without act ivation Sister chromatic exchange (SCE) in mammalian cells, with and without activation 4-25
1 TABLE 4-4 Organisation for Economic Co-operation and Development (OECD) Test Guidelines For detection of gene mutations: Salmonella/microsome assay with and without exogenous mammalian (S-9) enzyme activity Mammalian-cell point-mutation assay with and without exogenous metabolic activity For detection of chromosomal damage: In viva cytogenetics assay in rodents In vitro cytogenetics assays with and without exogenous metabolic activity: Sister chromatic exchange Chromosomal aberrations 4-26
- TABLE 4-5 Genetic Markers Developed in Cultured Mammalian Cells Resistance to cytotoxic chemicals--e."., 8-azaguanine (8-AGR), 6-thioguanine (6-TGR), 5-bromo-2'-deoxyuridine (BUdRR), a-amanitin, aminopterin, ouabain (OuaR), cytosine arabinoside, diphtheria toxin Glutamine or asparagine independence Auxotrophy (e.g. , adenine or praline dependence) Temperature sensitivity (TS) 4-27
TABLE 4-6 Markers for Evaluating Mutagenesis in Cultured Mammalian Gel Is Merits ~ _ Purine-analogue rests Lance Specificity: Low spontaneous background No lethal mutants, because non essent ial pathway involved Detects both base-pair and frame- shift alterations, with latter more efficient Dominance: X-linked 5-Bromodeoxyuridinea resistance Specificity No lethal mutants, because non- essential pathway involved Detects both base-pair and frame- shift alterations, with latter more efficient May detect chromosomal altera- tions Expression time: short Ouabain resistance Specificity: low spontaneous back- ground Dominance: independence of ploidy and genotype Artifacts: minimal aTrifluorothymidine also used. Limitations . Cross-feeding occurs; need for refeeding; use of special selection medium Influenced by ploidy; must be heterozygote, because auto- somal trait; need for preselec- tion of population before use Selective responsiveness (reacts only to ouabain mutagens-- base substitution); limited spectrum and frequency of mutants; no simple back- selection 4-28
TABLE 4- 7 Comparison of Properties of Some Ma~mnalian Transformation Systems S:ys tem Fischer rat emb ryo (F1706) Hams ter in vi tro co tony assay BALPs/3T3 clone A31 or C3H TOT 1/2 clone B Advant age s 1 . Shown to corre 1 a te wi th in viva test results in double-blind study 2. Easy to discriminate between normal and trans- formed morphology 3. Transformed ce 11 s do no t need to be cloned before inoculation into the syn- geneic host 1. High levels of mixed- function oxidase activity 2. Diploid chromosome complement 3. 4. Rapidity and reproduci- bility of test Characterized independ- ently in several laboratories . Can be used with meta- bolic activation 6. Quantitative results possible 1. Fairly rapid 2. Easy to discriminate between normal and trans- formed colonies 3. Well characterized by independent laboratories 4. Can be made quantitative 5. High correlation between phenotypic morphology and tumorigenes~s 4-29 Possible Disadvantages ' Not a cloned population ^. Only useful at certain passage levels 3. Low passage cells must be preinfected with a type "C" RNA virus . Aneuploid 5. Difficult to quantitate transformation 1. Low cloning efficiency 2. 3. Heterogeneous population Discrimination between normal and transformed morphologies somewhat subjective 4. Need for pretested, specific lots of fetal calf serum 5. Variation in sensitivity he tween d i f ferent embryo pools 1. Period of usefulness in tenms-of sensitivity to focal transformation by chemical carcinogens is unknown 2. Aneuploid 3 . Need for pretested, specific lots of serum 4. Intermediate level mixed- function oxidase activity
Table 4-7 (continued) System Advantages Possible Disadvantages , Human cells 1. May reflect more 1. Not well characterized accurately human in viva conditions 2 . Some Aye t ems require genetical ly aberrant 2. Diploid chromosome target cells compl emen t ~ s ome sys tems ~ 3. Experience necessary to recognize transformed phenotypes 4. Usually a very long latency period ? 5. Low levels mixed-function oxidase activity 4-30
TABLE 4-8 Mutagenic Activity of Various Particulate Emissions and of Cigarette-Smoke Condensates, Compared with Benzo~a~pyrenea Source of Emission Mutagenic Activity, revertants/ 100 fig of organic material Without S-9 With S-9 Spark-ignition engine138 342 Diesel engineb66 Unaltered 1,225 Unaltered Cigarette-smoke condensates0 - 98 Coke ovens164 252 Roofing tarO 99 Benzotaipyrene0 15,202 aData from Claxton,28 who used TA98 strains of with and without the S-9 activating systems. lmonella tyRhimurium bDifferent values were obtained for various diesel engines; the lowest and highest are given here. 4-31 ·?
TABLE 4-9 Influence of Alcohol on Direct Mutagenicity of Particulate Extracts of Spark-Ignition-Engine Exhausta Particulate TA 100 Revertants/ Emission, Revertants/ Vehicle and Fuel g of Extract mg/mi mile Ford Escort: Gasoline 10 1.5 15,000 Ethanol blendb 9 1.1 9,900 Gasohol 4 1.2 4,800 01 dsmob lie Cutlass: Gasoline 10 1.7 17, 000 Ethanol blendb 5 0.6 3,000 Gasohol 13 0.6 7,800 Chevrolet Citation: Gasoline 17 1.9 32,300 Ethanol blendb 14 0.9 12,600 Gasohol 10 1.0 10,000 Mercury Monarch: Gasoline 16 7.1 114,000 Ethanol blendb 12 2.8 34,000 Gasohol 20 1.1 44,000 aData from Naman and Clark.l19 Ego% gasoline-10% ethanol . ~ 4-32
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TABLE 4- 11 Bacterial Mutagenic Act ivity of PAHs in Kerosene-Soot Extracta Mutation Contribution, induced mutant fraction x 105 . . _ Weight ~Conc. Extract Conc. Extract in Extract = 20 ~g/mlb = 100 ~g/ml Compound Cyclopenta[cd]pyrene 15 30 l65C Pyrene 8 0 1.7 Benzo~ghi~perylene 8 2.6 3.4 and anthanthrene Coronene 5 0 105 Phenanthrene and anthracene Perylene 2 2 1.4 34 O O Benzo[a]pyrene and 1 0.6 3.4 benzote~pyrene Uncharacterized 18.3 Total CH2012 100 20 150 ,.. aData from Kadin _ al. JO bKadin_ al. determined the amounts of the individual PAHs in the kero- sene soot and, knowing the mutant fractions that these amounts would induce from a dose-response curve, were able to estimate mutagenic con- tributions that compounds would elicit. The induced mutant fraction = [(no. colonies exhibiting azaguanine resistance in presence of mutagen)/ (no. azaguanine-resistant colonies in absence of mutagen dilution factor). CBecause of nonlinearity of dose-response relationship, compounds may contribute differently to mutagenic response, depending on amount of soot extract and therefore on amount of individual PAR. 4-34
TABLE 4-12 Mutagenic Efficacy of PAHs in Relation to Benzo[a]pyrenea Concentration, Relative Mutagenic Compound~g/ml Potencyb 2-M~thylanthracene15.4 0.15 9-~ethyLanthracene14.4 0.08 1-Methylphenanthrene15.4 0.50 2-~ethylphenanthrene7.7 0.30 Pyrene28.3 0.07 1-Methylpyrene17.3 0.05 Cyclopen-ta[cdipyrene13.9 1.51 Benz~aianthracene14.8 0.14 Chrysene10.3 0.20 1,2-Benzodibenzothdithiophene117.0 -o Fluoranthene1.0 1.00 Benzota~pyrene1.3 1.00 Benzoteipyrene22.7 0.11 Perylene2.8 6.00 Anthanthrene12.1 0.08 Dibenz~ac~anthracene3.6 0.77 Dibenz~ahianthracene20.9 0.08 Coronene51.0 0 or Anthracene40.0 0 or Phenanthrene or Dibenzotbdithiophene v, aData from Kadin _ al.°~ 53.4 55.2 o bRelat~ve to benzo~a~pyrene, set at 1.00; rate-limiting factor is concentration that produced too much cell death. 4-35
TABLE 4-13 Mutagenicity of Nitrated Pyrenes in Salmonella typhimur~um TA 98 and TA 98 NRa CompoundTA 98b TA 98 NRb TA 98/TA 98 NR 1-Nitropyrene484 35 14 1,3-Dinitropyrene28,600 4,900 5.8 1,6-Dinitropyrene36,350 37,850 1.0 1,8-Dinitropyrene75,500 75,500 1.0 1, 3,6-Trinitropyrene31,400 28,220 1.1 1,3,6,8-Tetranitropyrene7,700 5,200 aData from Mermelstein _ al.112 1.5 bStrains TA 98 and TA 98 NR are nitroreductase-positive and -negative respectively. 4-36
TABLE 4-14 Mutagenicity of Sulfur-Containing Heterocyclesa No. Revertants Material Dose, fig (TA 98)b Occurrence Control Benzo~a~pyrene 36 0.5 78 2.0 277 Dibenzo~bd~thiophene 20.0 27 Tobacco smoke Phenanthrot4,5-bod]- O.S 73 Coal tar; thiophene 1.0 85 carbon black Benzotbinaphtho- 10.0 44 [2,1-dithiophene Renzo[2,3Jphenanthro- 0.5 122 Coke-plant t4,5-bedlthiophene effluent Triphenylenet4,5-bed]- 10 thiophene 42 Dinaphthot2,1-b;l', 10 36 2'-dithiophene aData from Karcher et al.87 bIn presence of activating S-9. 4-37 ~ . .. ..
TABLE 4- 15 Mutagenesis in CHO Cellsa Add i Lion Pos i t ive control--me thyl me thanesul fonate Diesel-exhaust particulate extracts Spark-ignition engine extract Coke-oven emis s ion Concentration Yielding Comparable Mutation Frequency ~ ~ u /ml 175 100-2 7 5 200 175 aResults extrapolated from data of Casto et al.22 CHO cells were treated with test agent at various doses for 16-24 h. Cells were collected, and 105 ce11s were inoculated into dishes. Mutant cells were selected for resistance to 6-thioguanine. bConcentration of test agent yielding mutation frequency of 5 x 105 CExtracts obtained from two different engines. 4-38 . ...
TABLE 4-16 Mutation Frequency of Test Agents with Bal/Bc 3T3 Cellsa Source Emission Extractb Mutation Frequen Without Activation With Activation Solvent control 0.18 0.26 Positive control, MING 35.5 - (1 ~g/ml) Diesel engine 0.18-1.06d 0.20-1.81 Spark-ignition engine 4.49 3.97 Roofing-tar pot sample 3.14 1.73 Coke oven 8.17 - Benzota~pyrene -- 14.2 (12.5 ~g/ml) Data from Curren et al.34 Particles were extracted with dichloromethane. Extract was used at seven concentrations in mutagenesis assay in absence of metabolic activation. Dose ranges included: diesel extract, 10-300 ~g/ml; roofing tar, 10-300 ~/ml; spark-ignition engine, 2.5-500 ~g/ml; and coke oven, 10-1,000 ~g/ml. Number of ouabain-resistant colonies per million viable exposed cells. Diesel exhausts from one heavy-duty and two light-duty engines are included; former yielded lower value. 4-39 id_ a.- .
TABLE 4-17 Induction of Ouabain- and 8-Azaguanine-Resistant Mutants by PAHsa Mutants/106 Survivors_ Cloning Treatmentb Efficiency, ~Resistant Resistant Solvent 92 1 6 Pyrene 94 1 5 Phenanthrene 79 1 8 Chrysene 85 2 9 Benz~ajanthracene 92 2 9 Dibenz~ac~anthracene 95 3 22 Dibenz~ah~anthracene 79 4 17 7-Methylbenz~a]- 61 24 75 anthracene Benzo~a~pyr~ne 27 45 - 128 (0.3 ~g/ml) 7,12-Dimethyl- - 50 22 41 benzanthracene (0.01 ~g/ml) 3-Methylcholanthrene 41 38 152 (0.3 ~g/ml) : aData from Hub erman and Sachs.78 ball compounds added at 1 ~g/ml unless otherwise stated. 4-40 arc
TABLE 4-18 Weight Proportion of Various PAHs in a Simulated "AEC" Mixturea Component Benzo[~]phenanthrene Cyclopentenopyrene Benz~aJanthracene Chrysene Benzotbifluoranthene Benzo~k~fluoranthene Benzo~j~fluoranthene Benzota~pyrene 1,12-Methylenebenzoteipyrene 10,11-Methylenebenzotaipyrene Dihenzo~aj~anthracene Indenot1,2~3-cd~pyrene Dibenz~ah~anthracene aData from Misfeld.ll4 I; 4-41 Weight, fig 0.08 1.85 O .09 0.21 0.17 0.06 0.09 0.30 0.14 0.05 0.10 0.21 0.02 ^. .. .. ..
TABLE 4- 19 Carcinogenic Activity of AEC, DEC, and PAHs on Mouse Skina Treatment, ~ g ~ Tumors Latency Period, wk Solvent `:ontrol o Benz ota]pyrene: 3.85 32.8 7 .69 60.9 74 61 15 .4 89 .1 44 AEC :b 290 10.3 72 880 44 .3 2, fi30 83 .3 72 52 DEC: b 4, 300 00 8, 600 2 .6102 l7, 150 12.7 Mixture of PAHs: I: aData from Misfeld. 114 bob ta ined wi th leaded fue 1 3.51.3 10 .538 .7 . CSee composition in Table 4-18. -~£ 4-42 76 91 73
TABLE 4-20 Carcinogenic Potency of Various Emissions and PAHsa Substanceb Benzota]pyrene Roofing-tar emission Coke-oven emission Caterpillar diesel exhaust Oldsmobile diesel exhaust Nissan diesel exhaust Potency, papillomas/mouse-mg A _ . 46 0.2 0.3 o 0.1 0.3 Mustang gasoline-engine exhaust 0.1 Cigarette-smoke condensate aData from Slaga et al.158 o bMaterial was applied to mice once as initiator. TPA (2 Egg, twice a week, was used as promoter. Amount of emission condensate that yielded linear response of tumors vs. dose was used. 4-43
Carcinogens Benzo[a]pyrene Dibenz~ahianthracene Benz[~]anthracene Benzo~blfluoranthene Noncarcinogens Phenanthrene Anthra`:ene Fluorantl,ene Pyr`ene Chrys ene Benz o[~]pyrene Benzotghi~pyrene Carcinogens + Noncarcinogens Carcinogens Nonearcinogens aData from Schmahl et al.149 TABLE 4-2 1 Mixtures of PAHs and Their Proportionsa Amounts, fig 2 3 1.0 1.7 3.0 0.7 1.2 2.1 1.4 2.4 4.2 0.9 1.5 2.7 Total 4.0 6.8 12.0 4 5 6 7 27.3 81.0 243.0729.0 8.5 25.5 76.5229.5 lO.8 - 32.4 97.2291.6 13.8 41.4 124.2372.6 1.2 3.6 10.832.4 0.6 1.8 5.416.2 3.1 9.3 27.983.7 Total 65.3 195.0 585.01,755.0 8 9 4.0 6.812.0 Total 65.3 110.5 195.0 - 69.3 117.3 207.0 4-44 ~.. .. ..
TABLE 4-2 2 Carcinogenicity of PAHs in Combinationa Treatment, pg Solvent . O Benzo ~ a ~ pyrene: 1 .0 14 1.7 ~28 3.0 56 Carcinogens: 4.0 36 6.8 68 12.0 71 Noncarc inogens: 6 5 1 195 585 1, 755 Papillomas + Carcinomas o 1 17 Carc inogens + noncarc inogens: 69 5 0 117.3 60 207 .0 70 aData from Schmahl et al.149 .^ 4-45 it. - ....
TABLE 4-23 Tumor-In i t fat ing Act iv i ty of C igaret te-Smoke Ingredient sa Relative Tumorigenic: Concentration, Compound Activityb ng/cigarette B~nzo~a~pyrene +++ 10-50 5-Me thylchrys ene +++ 0 . 6 Dibenz ~ ah ~ anthra`:ene ++ 40 Benzo~b] fluoranthene ++ ~30 Benzo ~ j ~ f luoranthene - ++ 60 Dibenz [a] acridine ++ 3-10 Indeno ~ 1, 2, 3-cd ~ pyrene + 4 Benz [a] anthracene aData from Hoffmann _ al. 72 bMouse-skin `:ar`: inogenes is e -_ + 4-46 40-70 ~.. .
TABLE 4-24 Summary of Carcinogenicity and Mutagenicity of PAHs in Various Emissions Compound Anthracene 2- or 9-Methylanthracene 1,2-, 1,3-, 1,4-, or 2,3 Dimethylanthracene 9,10-Dimethylanthracene Phenanthrene 1- or 2-Methylphenanthrene Fluoranthene 2- or Pyrene 1- or 2-Methylpyrene 1-Nitropyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene 1,3,6-Trinitropyrene 1,3,6,8-Tetranitropyrene Cyclopentated~pyrene Benz~aJanthracene (BA) 1-, 3-, 4-, 5-, or 11-Methyl-BA 2-Methyl-BA 6-, 7-, 8-, 9-, or 12-Methyl-BA 1,7-, 1,12-, 2,9-, 2,10-, 3,9-, 3,10-, 4,2-, 4,12-, 5,12-, or 8,11-Dimethyl-BA 4,5-, 6,7-, 6,8-, 6,12-, 7,8-, 7,11-, 7,12-, 8,9-, or 8,12 Dimethyl-BA 9,10- or 9,11-Dimethyl-BA Fluorene 9-Methylfluorene Acridine Anthanthrene Chrysene 1 -Me thyl ~:l~rys ene 2-, 3-, 4-, or 6-Methylchrysene 5-Methyl`:hrysene Benzo [b ~ f luoranthene Benzo ~ j ~ f luo-~-an thene Benzo ~ ghi ~ perylene 3-Methyl f luoranthene Carc inogenic Activitya. o o o 0/+ o o o + o o 0/+ o ++ 'O ++ o o o + 0/+ + ++ ++ ++ 4-47 Relative In Vitro Mutagenic Act ivi tyb Animal O O o O O + + Ba`:teria ++ + o + + ++ ++ +++ +++ ++++ +++ ++ ++ ++ +c ++d +d o + + + + + , ....
TABLE 4-24 ~ contd Compound Psenzo~k] fluoranthene Benzo~a~pyrene (Bta]P) 2-, 3-, 4-, 6-, 11 Methyl-B[a]P 5-Methyl-B[alP 8-Methyl-B[a]P 1,2-, 1,3-, 1,4-, 1,6-, 2,3-, 3,6-, 3,12-, or 4,5-Dimethyl B[a]P Benzo[~]pyrene Perylene 3-Methylcholanthrene Indeno[1,2,3-cd~pyrene Dibenz[ahianthracene (DBA) 2-, 3-, or 6-Methyl-DBA 7-Methyl-DBA Coronene Benz[aJacridine Dibenzo[bd~thiophene Dibenz~ac~anthracene Carcinogenic Activitya ++ ~+ ~ or 12 ++ + o ++ 0/+ o ++ + + + ++ o/+ + + In Vitro Mutagenic Activit b _ Y Animal Bacteria ++ ++ + + O ++ ++ ++ + ~ O O o + + 80, no tumors; +, tumors in up to 33% of animals; ++, tumors in over 337 of the animals. bBenzo~aipyrene mutagenicity set at ++ ~7-Methyl-BA. d7,12-Dimethyl-BA. . 4-48 ) ^. ... .
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5 EFFECTIVE BIOLOGIC DOSE In the class of polycyclic aromatic hydrocarbons (PAHs), there are several chemicals that are environmental pollutants; some are carcino- genic in experimental animals, and some are suggested to be carcino- genic in humans.160 In the body, they are enzymatically converted to reactive forms that bind extensively and covalently to cellular macro- molecules.63~80~15l~l82 The covalent binding of reactive metabolites of PAHs to DNA is considered to be an essential first step in PAH induction of neoplasia.63~80~84~128~151~182 The damaged DNA cannot be fixed and results in a mutation within the cell unless enzymatic repair occurs first. There are many phanmacokinetic and enzymatic processes involved before the formation of reactive metabolites of PAHs, which may ultimately form adducts with DNA. Thus, the concentration to which a person is exposed is probably not a good measure of the biologic dose that causes neoplasia7~6 ,149 or other PAH-induced toxicoses (see Chapter 4~. This chapter develops the theme that some degree of PAH metabolite-DNA adduct formation in the target tissue can be used as a measure of effective biologic dose. The effective biologic dose of a substance is a reflection of its absorption, distribution, metabolism (activation or detoxification), and excretion. In the case of an alkylating substance, such as a PAH, that dose can be measured directly on the basis of the amount of alkylated DNA, itself a reflection of adduct formation. If the accumulation of adducts in DNA is greater than the capacity of the tissue to repair such lesions accurately and greater than the capacity of the tissue to replicate its DNA, then the presence of adducts will be indicative of the effective biologic dose. The chapter begins with a brief discussion of the pharmacokinetics of PAHs. That is followed by a discussion of the metabolism of selected PAHs. The in viva formation and disappearance of PAH metabolite-DNA adducts are next reviewed in detail. Finally, there is a discussion of the possibility of using PAH metabolite-DNA adduct content as a measure of effective biologic dose for in vitro mutagenesis, initiation of carcinogenesis, and inhibition of replication and transcription. PHARMACOKINETICS Many phanmacokinetic and enzymatic processes are involved before a PAH reaches a target cell and is metabolized to reactive metabolites that interact with DNA and other cellular macromolecules free Figure 5-1~. The oxidative metabolism of PAHs is usually by cytochrome P-450, and the formation of excretable glutathione, glucuronide, and sulfate conjugates results in a very complex metabolic profile. Thus, pharma- cokinetic information that would enable one to construct mathematical 5-1
modern of the tissue distribution, metabolism, covalent binding to cel lular macromolecules , and excretion of PAHs and metabolites as func- tions of exposure dose are nonexistent. However, sufficient studies have been done to allow some general ization regarding absorption, tis- sue distribution, and elimination of PAHs (see Santodonato et al.,160 pp. 6-1 through 6-27~. Most of these studies have only followed radio- activity in various tissues, urine, and feces after administration of radiolabeled PAHs. PAHs are readily absorbed after administration by various routes and are then rapidly removed from the blood and distributed into a variety of boa: tissues. Kotin et al.l09 examined the radioactivity derived from I C-labeled benzota~pyrene (BaP) in various tissues of rats and mice after intravenous, subcutaneous, and intratracheal administration. The blood concentrations resulting from intravenous injection were hardly detectable after 10 min. Radioactivity was found in stomach, intestine, liver, kidney, lung, spleen, testis, myocardium, urine, and feces. The pattern of distribution was independent of route of administration, except that particularly high lung concentrations followed intratracheal administration. These workers did not examine fat or mammary gland. Other investigators have shown that nonmetabo- lized BaP,19 3-methylcholanthrene (3-MC),19~40 and dimethylbenz~a]- anthracene (DMBAJ19~58 accumulate and persist more in fat and mammary tissue than in other tissues. Some PAHs induce neoplasia in the mammary glands of rats. Rees et al.l57 examined the mechanisms by which BaP and other PAHs are absorbed from the gut. Accumulation of BaP in averted sacs of small intestine increased exponentially with incubation-medium concentration. The transport of BaP from the sac tissue to the inside medium was found to be proportional to the concentration in the sac tissue. Thus, if the capacity of other tissues to absorb BaP from extracellular fluid (and blood) is proportional to the concentration of BaP in the fluid, then accumulation in the tissues should also be proportional to intragastric concentration. For example, this relationship was observed in adipose and mammary tissue 18 h after oral administration of BaP. Rees et al. postulated-a mechanism of physical _ _ adsorption onto the intestinal mucosal surface and then passive diffusion into and through the intestinal wall. The proportional nature of the accumulation in the t issue can be accounted for by two phases of adsorption, one unilayer and the other multilayer. Even if tissue accumulation of PAHs is proportionally related to exposure dose, these results should not be overinterpreted. The situation is dynamic; the accumulation is transient, in that PAHs are rapidly metabolized and removed from the body. Rees _ al. observed that BaP disappeared very rapidly from the thoracic duct lymph. Moreover, PAR metabolite-DNA adduct content in various tissues is not linearly related to exposure dose (as discussed later). A relevant route of environmental exposure to PAHs is deposition in the lung of particles with PAHs on their surfaces. In general, the degree of resent ion of PAHs in the lung is a function of the size and 5-2
composition of the particles carrying them. Several investigators have shown that BaP retention by the lung is higher when it is adsorbed on particulate carbon 39~85 dust,l64 ferric oxide,85 aluminum oxide, 85 and talcl45 than when it is not; carbon-particle size affects BaP retention, but the size of particulate ferric oxide or aluminum oxide does not. 5 However, some recent studies have sug- gested that particulate adsorption of PAHs does not alter retention time in the lung or their distribution to other tissues. Adsorption on ferric oxide did not increase the retention time of BaP in hamster lung after intratracheal instillation.57 Pylev et al.l55 examined the clearance of intratracheally instilled BaP from the hamster lung; the disposition and clearance from liver, kidney, and blood; and excretion into feces and urine. BaP was instilled alone or adsorbed on asbestos or carbon black. Although these studies were limited in scope, it was found that the disposition of BaP from lung to other tissues, the rate of tissue clearance of BaP, and the pattern of BaP excretion were not altered by the introduction of BaP into the hamster either in free form or bound to particles. Obviously, more studies on rates of clearance from the lung and the later fate of particle-adsorbed PAHs are needed to clarify the effects of particle size and composition. However, it can be concluded that distribution to other tissues occurs after pulmonary exposure to particles on which PAHs are adsorbed. Elimination of PAHs in animals occurs mainly by excretion of con- jugated metabolites into the feCeS.4,23,109,161,162 There is so excretion of metabolites into the urine--approximately 10% in the study by Kotin et al.l09 Excretion into bile can be very rapid. For example, 6 h after intravenous injection of [3H]BaP, 60-70t of the tritium appeared in bile or conjugated metabolites.23 PAR clearance from an animal probably is not limited by metabolic rates or biliary clearance of metabolites, but rather is affected by the persistence of nonmetabolized compound in various tissues (such as fat, skin, and mammary gland) or perhaps by adsorption on particles. The pharmacokinetics of a PAH will be influenced by prior treatment with chemicals capable of inducing enzyme systems that metabolize it. Schlede et al.l61~162 have shown that pretreatment of rats with unlabeled BaP markedly increased the plasma-disappearance rate of a tritiated dose of the same compound given intravenously; the effect was especially marked during the first 5 min after the intravenous administration of the radiolabeled material, and increased clearance lasted for 6 h. This effect of pretreatment with the compound was paralleled by a lower concentration of [3H]BaP in brain, liver, and fatty tissues; similar but more variable results were observed in lung tissue. These influences of BaP pretreatment on a later intravenous dose of t3H]BaP were also observed when the radiolabeled compound was administered orally. 3-MC and DMBA pretreatment of animals produced comparable effects on the metabolic disposition and tissue content of radiolabeled BaP. Pyrene and anthracene pretreatment had little or no such effect on the in viva disposition of this compound, nor (id phenobarbital. In other studies, the biliary excretion Of [1 C]BaP
was shown to be increased by pretreatment with the unlabeled compound; however, no increase in excretion of the 14C-labeled metabolites of BaP into bile was observed after pretreatment with this compound. These findings suggest that conversion of BaP to its metabolites may be the rate-limiting step in its biliary excretion. METABOL I SM OF PAH s . . An organism's processing of xenobiotic chemicals is determined by their physical and chemical characteristics. Figure 5-2 summarizes the possible events leading to carcinogenesis in a cell exposed to a xenobiotic toxic chemical. After uptake, the cell may simply excrete the chemical unchanged, as is the case with some metals and apparently inert materials, such as asbestos. A toxicant may contain functional attachment groups, such as hydroxyl or ketone, that can be conjugated to deactivating moieties like glutathione or glucuronic acid by cytoplasmic transferase. If the toxicant is a PAH or other relatively stable molecule, it will be attacked by the microsomal monooxygenases and form an electrophilic intermediate, which can later be conjugated to a deactivating moiety, detoxified, and excreted. Once an activated electrophile is formed, it can readily attack nucleophilic sites other than the detoxifying substrates, such as nucleic acids and proteins. The formation of adducts between electrophile metabolites of PAHs and UNA is probably a necessary first step in the initiation of carcinogenesis by PAHs. The in vivo formation of PAH metabolite-DNA adducts is discussed later in this chapter. These biochemical changes to biologically active intermediates depend on the balance between enzyme systems: those enzymes generating and those detoxifying the intermediates. One of the major enzymes involved in activation is aryl hydrocarbon hydroxylase (AHH). It is found in virtually all eukaryotes (and some prokaryotes), has a wide range of specificities for substrate activity, uses a variety of iron-containing cytosolic pigments as the active sites for chemical oxida&~on (e.g., cytochrome P-450), and is substrate-induc- ible. ,134 Many PAHs are capable of inducing one or more forms of cytochrome P-450. There is some evidence that induction is regulated by one ~ene ~: a relatively small number of genes in animal-model systems 10,1 and perhaps even in humans (see Chapter 7~. The basis for genetic regulation appears to reside in a balance of inducers and receptors that are activated by PAH metabolites; after binding, trans- location to the nucleus, expression of induction-specific RNA, and protein s~nthesis, the generation of specific cytochrome P-450 is observed. In the murine-model systems, genetically controlled AHH activity is correlated with cancer formation caused by PAHs, such as BaP,1lO 3-MC,1l0 dibenz~aJanthracene~lll and DMBA.1l0 5-4
Examples of enzymes that can detoxify these metabolic intermediates are UDP-glucuronosyltransferase, glutathione-S-epoxide transferase, aryl sulfatase, and epoxide hydrase. These enzymes catalyze the conjugation of the primary oxidative species formed as a result of AHH activity to forms that are sufficiently polar to be excreted from cells and from the body. Some of the conjugating enzymes are also under a form of genetic control, 142 but their role in PAH carcinogenesis is not completely defined. Epoxide hydrase is one of the enzymes that had -been thought to function in a manner that results in the detoxification of PAHs; however, it is now established that, for a variety of PAHs, epoxide hydrase can catalyze the formation of dihydrodiol derivatives of PAHs and that these dials may serve as substrates for monooxygenase activity again--resulting in the formation of diol-epoxides.63 The diol-epoxides constitute at least one of the ultimate mutagenic and carcinogenic forms of PAHs. Over the last decade, BaP has been the most'exhaustively studied PAH carcinogen and has been the prototype compound in developing the mechanism of action of the cellular monooxygenase and cytoplasmic transferases necessary to activate and detoxify PAH carcinogens. A recent exhaustive summary of BaP metabolism dealt with its activation, carcinogenesis, and role in the regulation of mixed-function oxidases and related enzymes.63 A composite of metabolic products of BaP is shown in Figure 5-3. BaP has been studied in a large number of in viva and in vitro systems, as well as in cell-free preparations using homogenates, microsomal fractions, and purified enzymes. BaP may form epoxides at several sites around its ring system, and three epoxides (4,5-,7,8-, and 9,10-) have been identified. Research over the last half-decade has implicated the 7,8-diol (bay region*~94 as the primary precursor for the second round of activation by mixed-function oxidases, both cytoplasmic and nuclear,80 that form the highly electrophilic 7,8-diol-9,10-epoxide (Figure 5-4), which opens to form a trial carbonium intermediate. This reactive molecule has been shown to be the major species that binds to nucleic acids via the C-10 position of BaP and to exocyclic amino groups of guanine. Metabolism of many PAHs other than BaP has also been shown to proceed via diol-epoxides, such as benz~a]anthracene, l74'l88 chrysene~ll6,l89 dibenz[ah]anthracene,1 0 5-methylchrysene 81 7 ~ethylbenzanthracene (7-MBA)~35~127 DMBA,16~46$91~132~174 and 3~MCe 105, 179 The ease of formation of carbonium ions by these diol-epoxides parallels the observed biologic activity of the parent chemic'al~.115 Metabolic profiles on some PAHs other than BaP are available, and salient features of their metabolism are presented below. *The bay region is a molecular region between adjacent fused aromatic rings (see reference 115~. 5-5
BENZO[e]PYRENE Benzo~e~pyrene is a marginally carcinogenic structural isomer of the strong environmental carcinogen BaP. It contains two bay regions and, by theoretical calculations, should approximate BaP in carcino- genic activity. Metabolic studies have determined that the prob- able reason for its lack of carcinogenicity is that its major metabo- lism is distal to the bay region, so that the molecule does not favor formation of diol-epoxide intermediates. Its metabolism has been studied in hamster embryonic eel is and in eel 1-free preparations from rat liver. Its ma jar metabolize is 4,5-dihydro-4,5-dihydroxybenzo- [e ~ pyrene. Large-ecale experiments with microsomes positively identi- fied 9~10-dihydro-9,10-tihydroxybenzo~e] pyrene , but i t constituted le 8 8 than 1: of the total metabolites . ~1 "nzo 1 e ~ p~rrene ARC H . ~ 5-6 HO_~ H At loJ 4,5-Dlhydro-4,5- t thydroxy ~X?°H 9, lO~bydroxy
PYRENE Early studies on pyrene metabolism were in rats and showed increased urinary excretion of sulfuric acid esters and glucuronic acid conjugates. Later, 1-hydroxypyrene and 1 )6-dihydroxypyrene were identified.78 More definitive studies of pyrene metabolism were performed in rabbits and rats by analysis of urinary metabolites after intraperitoneal in Section. 172 No direct structural analysis was performed , but the resul ts o f a number o f chromatographic and spectral analyses were compared with synthetic s tandards. 1-Hydroxypyrene, 1,6- and 1,8-dibydroxypyrene, 4 ,5-dihydro-4 ,5-dihydroxypyrene , and N-acetyl- S-~4,5-dihydro-4-hydroxy-5 -pyrene ~ -L-c y s te ine were identified. The latter compound was also isolated from bile in rats. More recent studies with gas-liquid chromatography and mass spectrometry have con- firmed the presence of 1-phenolic and 1-dihydroxydihydro derivatives from rat-1 iver microsomal incubation and shown a marked increase in mutagenesis in the Salmonella T 90 and T 100 strains.79 '~3 :0H HO H 4, 5-Dihydro 4, 5 - ihydroxypyrene ~1 Colon ~OH N -acetylcystelne RE N7. f ~ 1 A NTHRACENE Pyrene ~ ~OH H O ~ 1, 8-D1 hydroxypyrene Benz[a]anthracene is a marginally carcinogenic PAN that has both bay-region and K-region areas. The original metabolic studies with benz~a~anthracene were done with thin-layer chromatography .21 ,26-28 ,75, 169, 171 (See reference 115 for explana~ Lion of the K-region. ~ 5-7 ~ ¢0H [me' O H O ~ I, 6-D1 hyd r oxypyrene
OH 3-tlydroxy 1 OHM; S-Bydroxy HO H on 8, 9-Dlhytso-8, 9~dltydroxy Benz 1e ]anthracene .; S. 6-Dthy dro-5, 6 , ~dlhydroxy These ~ tudies unequivoca 1 ly ident if fed 5, 6-dihydro-5, 6-d ihydroxybenz ~ a ~ - anthracene and 8,9-dihydro-8,9-dihydroxybenz~aJanthracene as major metabolites. Also reported were 1,2-dihydrodihydroxybenz~aJanthracene, 3-hydroxybenz~aJanthracene, 4-hydroxybenz~aJanthracene, and benz~a]- anthracene 7,12-quinone. In viva ~tudiesl9l with rats, rabbits, and mice reported a mercapturic acid derivative, presumably as a breakdown product of a cysteine conjugate. Also reported were trace amounts of 5-8 HO ~ SHOW A: _~- 1, 2-Dlhydro-1, 2- dthydroxy ~OH OH 3, 4-Blhydro-3, 4- dthydrosy
sulfate and glucuronic conjugates at the 3, 4, 8, and 9 positions, presumably as products of phenols; and 10,11-dihydro-10,11-dihydroxy- benz~ajanthracene were also reported. The 3,4-dihydro-3,4-dihydroxy derivative of benz~aJanthracene was later confirmed with high-pressure liquid Chromatography as a metabolite formed by rat-liver micro- somes.1 7 This 3,4-dihydrodiol adjacent to a bay region leads to the idea of benz~aJanthracene bay-region activation, including the possi- bility of an isolated double bond in the 1,2 position after formation of a diol-epoxide. That 3,4-dihydro-3,4-dihyroxybenz~aJanthracene is a minor product quantitatively, as opposed to the less active 5,6-diol, may explain the weak carcinogenicity of benz~aJanthracene. CHRYSENE ( 1, 2-BENZOPHENANTHRENE ) . This molecule is composed of two linearly annellated rings formed by pyrocondensation of carbonaceous malarial and is therefore present in coal tar in substantial quantities. Metabolism of chrysene has been studied in rodents and in cell-free and organ-culture systems. Incubation with rat-liver microsomes produced a series of hydroxylated OH Chrysene Hi; 1,2-Dihydro OH > ~5, 6-Dihydro ~OH 3, ~Dihydro 5-9
metabolizes, as seen by high-pressure liquid-chromatographic (HPLC) separation. Several of these metabolites have been identified with the use of synthetic standards.122 Three dihydrodiols have been char- acterized: the 1,2-, 3,4-, and 5,6-dihydrodiols. This metabolic profile has been concerned with the use of rat or mouse skin-organ culture.l23 The dihydrodiol metabolites are presumably formed through reactive epoxide intermediates by the P-450 mixed-function oxidases. However, no phenolic or quinoid structures have been identified from the remaining peaks in the HPLC separation.139 5-ME:THYLCHRYSENE Of all the methylchrysenes studied, only 5-methylchrysene shows any substantial carcinogenicity. Metabolism of this compound has been studied in the 9,000-g supernatant from rat liver.81~83 Liver homo- genates used for this work were prepared from Aroclor-treated male F-344 rats, and HPLC of metabolites showed nine peaks, of which seven had been identified (according to their relative abundance) as 5-hydroxy-5-methylchrysene, 5-methylchrysene 1,2-diol, 7-hydroxy-5- methylchrysene, 5-methylchrysene 9,10-diol, 9-hydroxy-5-methylchrysene, 1-hydroxy-5-methylchrysene, and 5-methylchrysene 7,8-diol. Two minor metabolites have not been identified. The bay-region theory would predict that 5-methylchrysene 1,2-diol and 5-methylchrysene 7,8-diol are primary candidates for active carcinogenic intermediates. However, experiments with liver homogenates indicated that formation of 5-methyl- chrysene 1,2-diol is favored over that of 5-methylchrysene 7,8-diol. No other biologic system has been used to study metabolism of 5-methyl- chrysene, so it is not possible to make any pertinent comparisons with other tissues or between intact-cell activation and detoxification. 5-10 ~. .... .