Jay Gandy

A review of the endocrine activity of parabens and implications for potential risks to human health.

Parabens are a group of the alkyl esters of p-hydroxybenzoic acid and typically include methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben, isopropylparaben, and benzylparaben. Parabens (or their salts) are widely used as preservatives in cosmetics, toiletries, and pharmaceuticals due to their relatively low toxicity profile and a long history of safe use. Testing of parabens has revealed to varying degrees that individual paraben compounds have weakly estrogenic activity in some in vitro screening tests, such as ligand binding to the estrogen receptor, regulation of CAT gene expression, and proliferation of MCF-7 cells. Reported in vivo effects include increased uterine weight (i.e., butyl-, isobutyl-, and benzylparaben) and male reproductive-tract effects (i.e., butyl- and propylparaben). However, in relation to estrogen as a control during in vivo studies, the parabens with activity are many orders of magnitude less active than estrogen. While exposure to sufficient doses of exogenous estrogen can increase the risk of certain adverse effects, the presumption that similar risks might also result from exposure to endocrine-active chemicals (EACs) with far weaker activity is still speculative. In assessing the likelihood that exposure to weakly active EACs might be etiologically associated with adverse effects due to an endocrine-mediated mode of action, it is paramount to consider both the doses and the potency of such compounds in comparison with estrogen. In this review, a comparative approach involving both dose and potency is used to assess whether in utero or adult exposure to parabens might be associated with adverse effects mediated via an estrogen-modulating mode of action. In utilizing this approach, the paraben doses required to produce estrogenic effects in vivo are compared with the doses of either 17β-estradiol or diethylstilbestrol (DES) that are well established in their ability to affect endocrine activity. Where possible and appropriate, emphasis is placed on direct comparisons with human data with either 17β-estradiol or DES, since this does not require extrapolation from animal data with the uncertainties inherent in such comparisons. Based on these comparisons using worst-case assumptions pertaining to total daily exposures to parabens and dose/potency comparisons with both human and animal no-observed-effect levels (NOELs) and lowest-observed-effect levels (LOELs) for estrogen or DES, it is biologically implausible that parabens could increase the risk of any estrogen-mediated endpoint, including effects on the male reproductive tract or breast cancer. Additional analysis based on the concept of a hygiene-based margin of safety (HBMOS), a comparative approach for assessing the estrogen activities of weakly active EACs, demonstrates that worst-case daily exposure to parabens would present substantially less risk relative to exposure to naturally occurring EACs in the diet such as the phytoestrogen daidzein.

ENDOCRINE MECHANISMS UNDERLYING REPRODUCTIVE TOXICITY IN THE DEVELOPING RAT CHRONICALLY EXPOSED TO DIETARY LEAD

A dose-response study was conducted in a rat model to examine the effects of lifetime lead exposure on the development of the reproductive system and the endocrine mechanisms underlying these effects. Time-impregnated female Sprague-Dawley rats (n = 10-15/group) were exposed to lead acetate in the drinking water at levels of 0.05%, 0. 15%, or 0.45% (w/v) initiated on gestational day 5. At birth, litters were culled to four male and four female pups. Exposure of dams to lead was continued until weaning, following which, the pups continued to be exposed to lead acetate in drinking water until sacrifice. One male and one female pup from each litter were sacrificed at age 21, 35, 55, and 85 d. A significant dose-responsive decrease in birth weight and crown-to-rump length was observed in all lead-exposed litters. However, no marked effects were observed on anogenital distance/crown-to-rump length ratios. Lead exposure resulted in a delay in sexual maturity as measured by prostate weight in male pups and time of vaginal opening in female pups, which increased with lead dose. These disruptions in reproductive physiology were accompanied by a significant decrease in neonatal sex steroid levels and suppression of the plasma concentrations of testosterone (male) and estradiol (female) during puberty. In male pups, this was accompanied by a significant decrease in plasma luteinizing hormone (LH), elevated pituitary LH content, and a decrease in plasma testosterone/LH ratios at the highest dose. In female pups, although no effects were observed on plasma LH concentration, a similar significant elevation in pituitary LH content was observed during early puberty. Postpuberty, plasma LH and sex steroid concentrations were unaffected at any dose in spite of continued lead exposure. No significant effects were observed on epididymal sperm count in male pups at 85 d of age. In female pups, estrus cycling was only significantly disrupted at the highest lead dose. These data suggest that the reproductive axis is particularly sensitive to lead during specific developmental periods, resulting in delayed sexual maturation produced by suppression by sex steroid biosynthesis. The mechanisms underlying this appear to involve lead actions on both LH release and gonadal function. At low, environmentally relevant blood lead concentrations, adaptation to the continuous presence of the metal ion occurs and surprisingly little effect is observed on adult reproductive endocrinology and physiology.

Reproductive toxicity: male and female reproductive systems as targets for chemical injury.

On the basis of current knowledge of reproductive biology and toxicology, it is apparent that chemicals affecting reproduction may elicit their effects at a number of sites in both the male and the female reproductive system. This multiplicity of targets is attributable to the dynamic nature of the reproductive system, in which the hypothalamic-pituitary-gonadal axis is controlled by precise positive and negative feedback mechanisms among its components. Interference by a xenobiotic at any level in either the male or the female reproductive system may ultimately impair hypothalamic or pituitary function. Normal gonadal processes such as spermatogenesis or oogenesis, ejaculation or ovulation, hormone production by Leydig or granulosa cells, and the structure or function of the accessory reproductive structures (e.g., epididymis, fallopian tube) also appear vulnerable to xenobiotics. The reproductive system is a complex one that requires local and circulating hormones for control. This brief review illustrates a system for characterizing the mechanism of action of reproductive toxicants, as well as for defining the sites available for disruption of reproduction. Unfortunately, at present, data addressing the actual vulnerability of reproduction are sorely lacking. However, when experiments have been conducted and combined with epidemiologic data or clinical observation, it has been possible to demonstrate impairment of reproductive processes by xenobiotics. The role of environmental exposure to xenobiotics in the increase in infertility that has been observed remains to be defined.

Glutathione depletion potentiates ethyl methanesulfonate-induced and damage to sperm chromatin structure

Male rats were treated with phorone at dosages previously shown to reduce glutathione in rodent reproductive tracts, followed by a single challenge with ethyl methanesulfonate, a known mutagenic and clastogenic agent. Epididymal sperm collected 8 and 15 days after exposure from phorone pretreated animals had a significantly greater alteration of sperm chromatin structure, defined as an increased susceptibility to DNA denaturation in situ, relative to sperm obtained from animals injected with saline alone or saline+EMS (50, 100, 150, or 200 mg/kg bw). These data support the hypothesis that ethyl methanesulfonate-induced alkylation of developing sperm chromatin protamines causes a significant stress on chromatin structure leading to increased DNA damage. This is the first report showing that glutathione depletion potentiates EMS-induced chromatin structural alterations that are likely related to dominant lethal mutations.

Comparison of the immunotoxicity of propanil and its metabolite, 3,4-dichloroaniline, in C57Bl/6 mice

Propanil (3,4-dichloropropionaniline), used extensively as a postemergence herbicide in rice and wheat, has as its major metabolite, 3,4-dichloroaniline (DCA). Propanil has previously been shown to affect the T cell-dependent antibody response. To determine the immunotoxicity of DCA, as well as extend the previous immunotoxicity studies, several T cell-dependent and -independent immune responses were determined after DCA or propanil exposure. Unlike propanil, DCA caused a significant reduction in T-dependent antibody production (anti-SRBC response) only at a high dose (150 mg/kg). DCA or propanil at 150 or 200 mg/kg, respectively, caused a significant reduction in the number of anti-DNP antibody producing cells. However, doses of 37 or 50 mg/kg of DCA or propanil, respectively, caused an increase in the number of anti-DNP antibody producing cells. These data indicate that both propanil and DCA have a differential effect on the T-independent antibody response depending on the dose. Similar to propanil, DCA (at 150 mg/kg) caused a significant increase in spleen weight and cellularity. The effect of DCA or propanil on selected cellular immune functions was also determined. DCA caused a significant decrease in the natural killer (NK) cell activity at doses of 75 or 150 mg/kg, and propanil caused a significant decrease at 100 or 200 mg/kg. Cytotoxic T lymphocyte activity, however, was unaffected even at 150 or 200 mg/kg DCA or propanil, respectively. Thus, it appears that T cells are relatively resistant to the effects of propanil and DCA, whereas, other immune cell types, e.g., NK cells are sensitive to its effects.

Effect of Acute Propanil Exposure on the Immune Response of C57BI/6 Mice

Propanil is a herbicide that is used extensively in rice farming to kill weeds without damaging the rice plant. The immunotoxic effects of acute exposure to propanil were determined in adult C57Bl/6 female mice exposed intraperitoneally to propanil at doses of 0, 10, 25, 50, 100, 200, or 400 mg/kg body wt. One week following exposure, the immune competency of the animals was assessed. Contact hypersensitivity response (CHR), blastogenic response to T- and B-cell-specific mitogens, and mixed lymphocyte reaction (MLR) were significantly depressed only in propanil-treated animals at 400 mg/kg. However, the number of splenic antibody-producing cells was also significantly depressed in a dose-dependent manner at the lower doses of 50, 100, and 200 mg/kg. In addition, a significant reduction in the thymus weight and an increase in absolute and relative spleen weight were also measured in animals treated with 200 and 400 mg/kg. The increase in spleen weight also showed a concomitant rise in spleen cellularity. These data indicate that propanil has a dose-dependent immunotoxic effect on the adult mouse that affects primarily the humoral response.

Cytotoxic T-lymphocyte and NK responses in mice treated prenatally with chlordane.

It has been reported previously that BALB/c mice, treated in utero with chlordane, showed increased survival to influenza A/PR/8/34 [H1N1] (influenza) virus as young adults. To determine the possible role of cell-mediated immunity (CMI) on this effect, cytotoxic T-lymphocyte (CTL) and natural killer (NK) cell activities were assessed on chlordane-exposed offspring at 100 and 200 days post partum. The CTL response of these offspring showed no significant change from that obtained from their sex- and age-matched control counterparts exposed prenatally to the vehicle. NK responses of chlordane-exposed female offspring were significantly higher at 100 days of age but not at 200 days of age. Although male offspring that were exposed to chlordane prenatally showed no difference in NK cell activity at 100 days of age, NK cell activity was significantly less in chlordane-treated animals than controls at 200 days of age. Thus, prenatal treatment of mice with chlordane had varying effects on the NK cell activity of adult offspring, depending on the sex and age of the animal. It is concluded that the previously reported increase in survival to influenza is due to a resolution of the infection by normal CTL and NK cell activities coupled with a decrease in delayed-type hypersensitivity (DTH)-mediated pathology.

Selective Myelotoxicity of Propanil

Propanil, a commonly used herbicide, has been previously shown to be immunotoxic for selected immune functions as well as specific cell types, such as the macrophage. Propanil has also been shown to cause a methemoglobulinemia and anemia through direct action on the erythrocyte. Demonstrated toxicity to both macrophages and erythrocytes raised concern for the possible myelotoxicity of propanil which could contribute to the observed effects of exposure. Therefore, the effect of propanil on several stem and progenitor cell types was assessed 7 days after acute propanil exposure. The results described herein show that propanil, at doses of 50-200 mg/kg body wt, resulted in reduction in the number of myeloid stem cells and early myeloid and erythroid progenitor cells. No reduction in the numbers of more differentiated myeloid and erythroid progenitor cells was noted at even the highest dose used (200 mg/kg). In addition, no statistically significant difference in number of leukocytes per femur was noted. These data suggest that propanil is myelotoxic to early hemapoietic stem cells, but that this reduction is apparently compensated by proliferation of more differentiated progenitor cells for the myeloid and erythroid lineages. It remains unknown whether chronic exposure leads to progressive depletion of additional myeloid and erythroid cells.

Effects of selected chemicals on the glutathione status in the male reproductive system of rats

Previous studies have suggested a significant role for reproductive tract glutathione in protecting against chemical-induced germ-cell mutations. Therefore, a number of compounds were tested for their ability to perturb glutathione levels in the testes and epididymides as well as liver following single acute dosages to rats. Phorone (250 mg/kg), isophorone (500 mg/kg), and diethyl maleate (500 mg/kg) significantly reduced glutathione in the liver and in both reproductive organs examined. Methyl iodide (100 mg/kg), trimethyl phosphate (600 mg/kg), naphthalene (500 mg/kg), acetaminophen (1500 mg/kg), and pentachlorophenol (25 mg/kg) affected hepatic and epididymal glutathione, but had little or no effect on testicular levels. The ability of isophorone to enhance the covalent binding of tritiated ethyl methanesulfonate (3H-EMS) to spermatocytes was assessed. Perturbation of reproductive tract glutathione by isophorone treatment significantly enhanced the extent of 3H-EMS-induced binding to sperm heads. The temporal pattern of ethylations in sperm heads was consistent with the stage of sperm development known to be susceptible to ethylations by EMS. Therefore, chemical-induced lowering of glutathione in the male reproductive tract may be a mechanism for potentiation of chemical-induced germ-cell mutations.

Changes in primary and secondary lymphoid organ T-cell subpopulations resulting from acute in vivo exposure to propanil.

Acute exposure to the herbicide propanil is immunotoxic for selected immune functions, as well as causing changes in the weights of the thymus and spleen. Although spleen cellularity and weight increase with propanil exposure, the thymus: body weight ratio decreases with increasing doses of propanil. The present study analyzes the thymocyte subpopulations in the thymus, spleen, and mesenteric lymph nodes. C57Bl/6 mice were treated with either 0, 100, 150, or 200 mg/kg propanil, and 7 d later thymocyte populations were analyzed by flow cytometry. In the thymus, propanil exposure resulted in a dose-dependent decrease in total numbers of T cells, as would be expected with its reduced weight. Determination of the thymocyte subpopulation distribution in the thymus showed a significant reduction in the number of CD3+CD4+CD8- (CD3+4+8-), CD3+CD4-CD8+ (CD3+4-8+), and CD3+CD4+CD8+ (CD3+4+8+) cells. Percent distribution of these thymic cell subpopulations showed similar decreases only with the highest dose. Apparent dose-related decreases in the numbers of CD3-CD4+CD8+ (CD3-4+8+) cells were also noted and were attributed to the general decrease in total thymus cells. The percentage of CD3- subpopulations showed an increasing trend with dose, which suggests that at 7 d postpropanil exposure there may be a specific effect on this most immature population. Although the size and cellularity of the spleen were increased, no change in CD4+ or CD8+ cell distribution was observed. Similarly, mesenteric lymph nodes showed no changes in the cell subpopulation distribution between propanil-treated and control animals.