Drinking Water and Health, Volume 6 (1986) / Chapter Skim
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3. Reproductive Toxicology
3. Reproductive Toxicology
Pages 35-104
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From page 35... ...
For example, chromosome abnormalities detected in the embryo can arise from lesions in the germ cells of either parent before conception or at fertilization, or from direct exposure of embryonic tissues during gestation. Major malformations, however, usually occur when exposure occurs during a discrete period of pregnancy, extending from the third to the eighth week of human development.
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From page 36... ...
Consequently, the bulb of the information on specific exposures reported to affect reproductive function is derived from animal studies. The standard toxicological testing procedures for acute, subacute, and chronic exposures are not appropriate for detecting reproductive effects either in humans or in animals.
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From page 37... ...
Other areas not as extensively studied are toxicities affecting the male and female reproductive systems, resulting in sexual dysfunction and infertility. Direct damage to germ cells, neuroendocrine imbalances, and alterations in accessory reproductive organs, which can be involved in these toxicological processes, are described in the following sections as they have been elucidated in animal studies.
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From page 38... ...
Extensive physiological degeneration of germ cells occurs during the oogonial and primary oocyte stages of development. In humans, an estimated 70% of the germ cells present in a 5-month-old fetus are lost before birth (Biggers, 19801.
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From page 39... ...
Adapted from Tsafriri, 1978. synchrony, which accounts for the majority of germ cell loss.
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From page 40... ...
During the prepubertal and reproductive periods, the majority of germ cells remain as primary oocytes enclosed within unilamellar follicles. These resting follicles comprise the pool from which a select number of oocytes are recruited for further maturation to preovulatory or graafian follicles.
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From page 41... ...
Oocyte Toxicity The ovary, as a repository of oocytes and as a source of steroid hormones that control the functional development of reproductive organs, plays a major role in fertility and initiation of pregnancy. As indicated in the preceding section, when folliculogenesis is complete in the female during the perinatal period, oogonial cells no longer persist.
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From page 42... ...
Effects of Radiation on Oogenesis In rodent species, female germ cells are extraordinarily sensitive to killing by exposure to ionizing radiation, especially during neonatal life. Primordial, or resting, follicles in juvenile mice have an LDso of only 6 reds (Dobson and Felton, 1983)
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From page 43... ...
In irradiated preovulatory oocytes, the incidence of dominant lethal mutations is highest at the first metaphase, slightly less at the second, and low at other stages (Baker, 19781. Effects of Xenobiotic Compounds on Oogenesis POLYCYCLIC AROMATIC HYDROCARBONS (PAHS)
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From page 44... ...
These agents destroy rapidly dividing granulosa cells in growing follicles as well as in the resting primordial follicle. When prepubertal girls are treated with antineoplastic drugs, complete loss of germ cells is unlikely.
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From page 45... ...
component of the female reproductive system functions in a permissive, integrating role. Hypothalamic neurons synthesize and secrete gonadotropin-releasing hormone (GnRH)
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From page 46... ...
It has been well established that exposure of female rodents to pharmacological doses of androgens or estrogens during fetal or neonatal life results in disruption of the mechanisms that control cyclic secretion of gonadotropins (Kraulis et al., 1978~. In addition, several contaminants in drinking water, such as Kepone, exhibit estrogenic activity and disrupt control mechanisms (Hudson et al., 19841.
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From page 47... ...
Insofar as aromatization of androgens to estrogens is not involved in masculinization of the primate hypothalamus, exposure to estrogenic agents is not likely to lead to abnormal patterns of gonadotropin release or male infertility. The primate hypothalamus appears to be insensitive to androgens; pharmacological exposure of female fetuses to androgens can masculinize the external genitalia without influencing the periodicity of the adult menstrual cycle (Clark, 1982)
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From page 48... ...
We can conclude, therefore, that exposure to steroid hormones during the perinatal and adult periods is associated with a number of reproductive tract abnormalities in females. Moreover, although there may be differences in the critical period and in the mechanism of action between rodent and primate species, pharmacological exposure to sex steroids early in life predisposes the adults of all mammalian species to subsequent effects on their reproductive system (Clark, 19821.
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From page 49... ...
At blood THC levels comparable to those found in regular human marijuana users, nonhuman primates experienced disruption of the menstrual cycle and inhibition of ovulation (Asch et al., 1981~. An 18-day exposure to THC resulted in disruption of the menstrual cycles that persisted until 6 months after treatment (Asch et al., 1979~.
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From page 50... ...
Juvenile male rats receiving an identical treatment regimen during sexual maturation had severalfold higher elevations in hormone levels after withdrawal than did adult male rats, and the period of elevation persisted for 80 days. Other tranquilizers known to alter the hypothalamic GnRH level through effects on endogenous catecholamines are reserpine, chlorpromazine, and perphenazine (McLachlan et al., 19811.
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From page 51... ...
In these studies, animals are continuously exposed to the test compound, usually in the food or drinking water, for three generations. A new test procedure entitled Fertility Assessment by Continuous Breeding (FACB)
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From page 52... ...
_ Measurement of Hormone Patterns FIGURE 3-4 Flow chart for National Toxicology Program Fertility Assessment by Continuous Breeding protocol. From Lamb et al., 1984, with .
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From page 53... ...
Maturation of the Male Reproductive System In the early fetal period, prespermatogonial germ cells undergo mitotic proliferation and migration to the gonad, as described earlier in this chapter
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From page 54... ...
As a direct consequence of androgen synthesis in the fetal testis, the male reproductive tract grows and differentiates, and the accessory glands and external genitalia are formed (Gondos, 19801. A major difference in male and female germ cell development is that male germ cells do not enter meiosis until puberty.
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From page 55... ...
Sperm Toxicity An estimated cell loss of 35% occurs from the early spermatocyte to the spermatid stage due to physiological atresia (Salisbury et al;, 19771. Unlike germ cells in the female, however, cells lost to atresia in the male can be replaced by continual production of spermatocyte cells from division of stem cells.
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From page 56... ...
FSH also influences the biosynthesis and interconversions of steroid hormones (Phillips et al., 19851. Effects of Radiation on Spermatogenesis The effects of radiation on spermatogenesis have been studied in a variety of species with different model systems and indices of measure
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From page 57... ...
Test Systems for Evaluating Mate Reproductive Toxicity in Laboratory Animals ENTRY-LEVEL TESTING The study of male reproductive toxicity encompasses scientific approaches ranging from general toxicity testing to examination of the mechanism of action. The first step in assessing the potential action of an agent on male reproductive function should be to conduct tests in the intact animal so that the spectrum of physiological processes involved can be examined at one time, regardless of the target organ, cell, or molecule.
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From page 58... ...
Furthermore, the percentages of progressively motile sperm and of morphologically normal sperm in human semen are lower than those typical of males in other species. Consequently, measures of fertility and fecundity in animal models may be insensitive criteria for monitoring the integrity of the male reproductive system, and for identifying agents with the potential for causing reproductive toxicity in men (Amann, 19821.
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From page 59... ...
If an elevation in epididymal sperm with morphological abnormalities was identified, for example, secondary testing could be conducted to identify germ cell mutations, which is described later in this chapter. Similarly, agents that did not affect sperm production but that caused failures in mating could be evaluated for effects on the physiology of ejaculation.
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From page 60... ...
Fathers of 201 spontaneous abortions had significantly more sperm shape abnormalities and lower sperm counts than did fathers of 116 normal pregnancies. Although several studies support a link between sperm defects and abnormal reproductive outcome, others found no correlation (see Wyrobek et al., 1984, for a review)
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From page 61... ...
Selective pressures against sperm with grossly imbalanced chromosome complements and abnormal morphology are exerted during spermatogenesis and in the female reproductive tract. If genetically damaged sperm do fertilize an egg, a major selective pressure is embryo death; relatively few if any embryos carrying the genetic damage survive to term.
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From page 62... ...
Mitochondria Soiral Ring Centriole abnormalities in determining the nature of the offspring will be important, because semen analysis is particularly adaptable to human reproductive risk assessment. As this method gains wide acceptance, it will be necessary to determine how predictive it is of male reproductive insult versus the potential for adverse pregnancy outcome.
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From page 63... ...
Methods for analyzing semen from humans and laboratory animals have undergone extensive development in the past 5 years. The evolution of basic research in mammalian sperm physiology has resulted in several new techniques for assessing human sperm functions.
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From page 64... ...
This quality of fluorescence seems to be unique to the Y chromosomes of humans and the higher apes. Nonetheless, this system may provide a useful model of chemically induced nondisjunction in male germ cells.
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From page 65... ...
, and it is uniformly negative in mammalian germ cell mutation assays (Epstein et al., 19721. A variety of pharmacokinetic and adaptational factors in germ cells are probably the cause of this apparent mutagenic selectivity.
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From page 66... ...
Nevertheless, an indication of mutagenic activity in the germ cells of test animals must be taken as a warning of potential mutagenic activity in humans. The most relevant animal models are those in which germ cell mutations can be measured directly by the heritable translocation test and the specific locus test.
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From page 67... ...
At present, sperm-oocyte interaction tests are most appropriately applied in settings such as artificial insemination clinics rather than in surveillance studies of human populations. EXAMINATION OF SEMINAL FLUID The average ejaculate from a fertile man comprises approximately 3 ml of semen containing 50 million to 120 million sperm per milliliter of seminal plasma.
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From page 68... ...
The biochemical composition of the seminal plasma is complex and differs in many respects from that of blood plasma and other body fluids. The measurement of components in seminal fluid specific to the secretions of each accessory gland in the male reproductive system can aid the clinician in evaluating the functional status of those glands.
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From page 69... ...
This potential association is of particular concern when chemical exposure occurs through contaminated drinking water, since exposure may be chronic throughout the reproductive years. Test Systems for Germ Cell Mutations Two types of mutagenic damage can be examined in germ cells: macrolesions and microlesions.
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From page 70... ...
. Heritable numerical chromosome anomalies result from missegregation of chromosomes during mitotic or meiotic divisions of the germ cells.
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From page 71... ...
The findings from studies in laboratory animals do, therefore, show that germ cell mutations induced by chemical exposure can result in passage of genetic diseases to the offspring. The dominant lethal, heritable translocation, and specific locus tests are the methods most commonly used in such studies to assess the mutagenic potential of chemicals in
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From page 72... ...
72 so Cd As: 2 ._ to Cal ._ Cal us 2 m Cal o Cal Cd Cal Us ._ Cal an ._ V)
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From page 73... ...
Therefore, male rats are conventionally bred on a weekly basis for 10 consecutive weeks after exposure. This timing permits sampling of germ cells exposed in the mature sperm, spermatid, spermatocyte, spermatogonia, and stem cell stages of maturation.
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From page 74... ...
74 o Cal o x no Cq Cal Cal s°Ct Cal is: Cal .= ._ Cal ._ o ~ Cal 4_ Cal _ 1 ~ ,~ no m EM Cal 4)
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From page 78... ...
The increased sensitivity of male germ cells to dominant lethal or other types of mutations is based on the fact that many mutations occur predominantly in replicating cells. As indicated in Table 3-7, the stem cell spermatogonia of male mammals continue to replicate throughout the breeding life, which confers susceptibility to replication-dependent mutations.
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From page 79... ...
Each of these would be expected to reduce the mutagenic response of germ cells relative to that of somatic cells. The important application of these assays is to determine whether an agent that causes genetic toxicity in somatic cells also causes heritable mutations in germ cells, or if testicular lesions or reductions in male fertility identified in reproductive toxicology studies are due to germ cell mutations.
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From page 80... ...
The levels of dominant lethal and heritable translocation mutations induced in male germ cells by x rays, by several direct-acting alkylating agents' and by benzotalpyrene are listed in Table 3-8. For a number of mutagens, including ethyl methane sulfonate, triethylenemelamine, and x rays, there is a positive correlation between the induction of dominant lethal mutations and the occurrence of heritable translocations.
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From page 81... ...
Thus, some compounds can induce both types of genetic changes, whereas others induce only one (Generoso, 19821. SPECIFIC LOCUS TEST As discussed above, the dominant lethal and heritable translocation assays measure dominant mutations that are macrolesions in the chromosomes of germ cells.
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From page 82... ...
, there is concern that the acceptable daily exposure level estimated from the subchronic study may be too high with respect to effects on reproductive function. Koeter evaluated toxicity data on 37 compounds tested both in subchronic studies and in one or more reproductive toxicity studies to determine the impact of the latter in identifying the NOEL and the LOEL.
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From page 83... ...
First, weanling animals (usually rats or mice, 30 to 40 days of age) are randomly assigned to control or test groups, and the test compound is administered continuously in the diet or drinking water or via inhalation for the duration of the study.
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From page 84... ...
When rodents are used, enough females should be started at each dose level so that at least 20 pregnant females per dose level for each generation are obtained. All doses are reported as mg/kg body weight per day, which necessitates measurement of food and water intake.
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From page 85... ...
In addition, it provides for multiple, sequential breeding (up to five mating cycles) of one parental generation, which permits identification of latent toxic effects on immature germ cells (resting follicles, spermatogonia)
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From page 86... ...
If males are identified as the affected sex, they are killed and bled by cardiac puncture. The following observations are then made and evaluated: body weight, liver weight, fixed brain weight, fixed pituitary weight, right testis weight, ventral prostate weight, seminal vesicle weight, right epididymal weight, number of cauda epididymal sperm per milligram of tissue, and sperm morphology and motility.
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From page 87... ...
The following end points are evaluated in task 4: number and percentage of fertile pairs, litter size, litter weight, and number and percentage of live-born animals. SEGMENT ~ STUDIES: GENERAL FERTILITY AND REPRODUCTIVE PERFORMANCE Segment I studies can be performed with exposures of males alone, females alone, or both, depending on the background information available.
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From page 88... ...
The intermediate dose should lie logarithmically between the lowest and highest level. TIME OF VAGINAL OPENING IN THE RAT PUP This test can be used to identify estrogenic activity of test agents such as the estrogen agonists DES, clomiphene, and tamoxifen, which cause premature vaginal opening in the rat.
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From page 89... ...
Relative changes in sperm production rates can be established by direct comparison of testicular spermatid reserves, i.e., the number of spermatids per testis, per pair of testes, or per unit weight of testicular parenchyma. The technique for determining testicular spermatid reserves involves homogenization of testicular tissue and subsequent enumeration of elongated spermatids by hemacytometry.
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From page 90... ...
These values are divided by 6.10 days (the time required for spermatids to form spermatozoa in the rat) to obtain the daily sperm production rate.
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From page 91... ...
The sperm specimen must be held at 37°C during taping in an air-curtain incubator. HISTOLOGICAL EVALUATION OF THE TESTIS The choice between histological or homogenization techniques for quantifying relative changes in sperm production depends upon the nature and severity of anticipated effects.
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From page 92... ...
1982. Effects of chemicals on chromosome-aberration production in male and female germ cells.
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From page 93... ...
1978. Alterations of germ cells leading to mutagenesis and their detection.
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From page 94... ...
1978. Vulnerability of female germ cells in developing mice and monkeys to tritium, gamma rays, and polycyclic aromatic hydrocarbons.
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From page 95... ...
1980. Induction of gene mutations in germ cells of the mouse.
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From page 96... ...
1978. Inducibility by chemical mutagens of heritable translocations in male and female germ cells of mice.
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From page 97... ...
II. Female Reproductive System, Part 1.
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From page 98... ...
1984. Reproductive toxicity of ethylene glycol monoethyl ether tested by continuous breeding of CD-1 mice.
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From page 99... ...
1979. Ovarian aryl hydrocarbon hydroxylase activity and primordial oocyte toxicity of polycyclic aromatic hydrocarbons in mice.
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From page 100... ...
1983. ~y-Ray-induced mutations in male germ cells of a recombinationdefective strain (c3G)
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From page 101... ...
1974. Assessment of the mutagenic properties of diquat and paraquat in the murine dominant lethal test.
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From page 102... ...
1978. Daily sperm production and epididymal sperm reserves of pubertal and adult rats.
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From page 103... ...
1975. Dominant lethal mutations and antifertility effects of di-2-ethylhexyl adipate and diethyl adipate in male mice.
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From page 104... ...
1978. Effects of methyl methanesulfonate and dimethylnitrosamine on sperm production in Syrian hamsters.
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