Daniel Desaulniers

Effects of mixtures of polychlorinated biphenyls, methylmercury, and organochlorine pesticides on hepatic DNA methylation in prepubertal female Sprague-Dawley rats.

DNA methylation is one of the epigenetic mechanisms that regulates gene expression, chromosome structure, and stability. Our objective was to determine whether the DNA methylation system could be a target following in utero and postnatal exposure to human blood contaminants. Pregnant rats were dosed daily from gestation day 1 until postnatal day 21 with 2 dose levels of either organochlorine pesticides (OCP; 0.019 or 1.9 mg/kg/day), methylmercury chloride (MeHg; 0.02 or 2 mg/kg/day), polychlorinated biphenyls (PCBs; 0.011 or 1.1 mg/kg/day), or a mixture (Mix; 0.05, or 5 mg/kg/day) including all 3 groups of chemicals. Livers from 1 female offspring per litter were collected at postnatal day 29. Hepatic analysis revealed that the mRNA abundance for DNA methyltransferase (DNMT)-1, -3a, and -3b were significantly reduced by the high dose of PCB, that the high dose of MeHg also reduced mRNA levels for DNMT-1, and -3b, but that OCP had no significant effects compared with control. The high dose of PCB and Mix reduced the abundance of the universal methyl donor S-adenosylmethionine, and Mix also reduced global genome DNA methylation (5-methyl-deoxycytidine/5-methyl-deoxycytidine + deoxycytidine). The latter is consistent with pyrosequencing methylation analysis, revealing that the high-dose groups (except OCP) generally decreased the methylation of CpG sites (position -63 to -29) in the promoter of the tumor suppressor gene p16INK4a. Overall, these hepatic results suggest that the DNA methylation system can be affected by exposure to high doses of blood contaminants, and that OCP is the least potent chemical group from the investigated mixtures.

Interactions between endosulfan and dieldrin on estrogen-mediated processes in vitro and in vivo

There is growing concern that estrogenic chemicals, both natural and human-made, may be causing a variety of reproductive disorders in wildlife and human populations. Recent in vitro data suggest that the interaction between some weakly estrogenic organochlorines, dieldrin, endosulfan, toxaphene, and chlordane, causes a synergistic increase in their estrogenic potency, an effect due to joint action on estrogen receptors (ER). As these studies were conducted using models of estrogen action derived from cells that are not physiologically controlled by estrogens, the relevance of these findings to human health are not clear. The present studies were conducted to examine the interaction between endosulfan and dieldrin in the activation of ER in or extracted from mammalian cells. Endosulfan and dieldrin showed no synergism in displacing 3H-E2 from rat uterine ER or in inducing the proliferation of MCF-7 breast cancer cells, an estrogen-dependent response. Furthermore, endosulfan (0.1 mg per animal per d) or dieldrin (0.1 mg), alone or in combination, injected intraperitoneally daily for 3 d, did not stimulate any uterotrophic activity nor had any effect on pituitary prolactin or other endocrine-related endpoints in immature female rats. These studies demonstrate that these weakly estrogenic compounds do not interact in a synergistic fashion in binding to ER or in activating ER-dependent responses in mammalian tissues or cells. Thus, these results suggest that coexposure to these weakly estrogenic environmental contaminants likely will not cause human reproductive toxicity related to estrogen action.

Causes of genome instability: the effect of low dose chemical exposures in modern society

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genomes integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Reproductive and Thyroid Hormone Levels in Rats Following 90-Day Dietary Exposure To Pcb 28 (2,4,4'-Trichlorobiphenyl) or Pcb 77 (3,3',4,4'-Tetrachlorobiphenyl):

Subchronic exposure to the PCB congener 77 (PCB 77) and 28 (PCB 28) was previously shown to induce histological changes in the thyroid and in the brain biogenic amines levels, suggesting possible effects on thyroid and reproductive hormone levels. Thus, the effects of a 90-day dietary exposure to PCB 28 or 77 on luteinizing hormone, follicle-stimulating hormone, and testosterone concentrations were studied in male rats, as well as the levels of thyroid-stimulating hormone, thyroxine (T4) and uridine diphosphate-glucuronyl transferase (UDP-GT) activity in both genders. Weanling Sprague Dawley rats were randomly distributed into groups of 10 rats and were fed, for the next 13 weeks, purina lab chow containing 50, 500, 5000, or 50 000 ppb of PCB 28 or 10, 100, 1000, or 10 000 ppb of PCB 77. The serum concentrations of T4 were decreased in rats of both sexes receiving 1000 ppb or more of PCB 77, and was associated with an increased activity of UDP-GT which reached significance only in the females. There was a tendency for the highest dose of PCB 28 also to decrease serum T4 concentrations in the female rats. None of the PCB treatments significantly altered gonadotropin, TSH, or testosterone concentrations. These results suggest that thyroid functions may be more susceptible or adapt less readily than the pituitary gland and the testes to endocrine disruption caused by PCB congeners.

Effects of postnatal exposure to a mixture of polychlorinated biphenyls, p,p'-dichlorodiphenyltrichloroethane, and p-p'-dichlorodiphenyldichloroethene in prepubertal and adult female Sprague-Dawley rats.

The postnatal period is a critical phase of development and a time during which humans are exposed to higher levels of persistent organic pollutants (POPs), than during subsequent periods of life. There is a paucity of information describing effects of postnatal exposure to environmentally relevant mixtures of POPs, such as polychlorinated biphenyls (PCBs), p,p′-dichlorodiphenyltrichloroethane (DDT), and p,p′-dichlorodiphenyldichloroethene (DDE). To provide data useful for the risk assessment of postnatal exposure to POPs, mixtures containing 19 PCBs, DDT, and DDE were prepared according to their concentrations previously measured in the milk of Canadian women, and dose-response effects were tested on the proliferation of MCF7-E3 cells in vitro, and in vivo experiments. Female neonates were exposed by gavage at postnatal days (PNDs) 1, 5, 10, 15, and 20 with dosages equivalent to 10, 100, and 1000 times the estimated human exposure level over the first 24 days of life. The MCF7-E3 cells showed a 227% increase in the AlamarBlue proliferation index, suggesting estrogen-like properties of the mixture, but this was not confirmed in vivo, given the absence of uterotrophic effects at PND21. An increase (511%) in hepatic ethoxyresorufin-o-deethylase activity at the dose 100× was the most sensitive endpoint among those measured at PND21 (organ weight, mammary gland and ovarian morphometry, hepatic enzyme inductions, serum thyroxine and pituitary hormones). In liver samples from older female rats (previously involved in a mammary tumor study [Desaulniers et al., Toxicol. Sci. 75:468–480, 2001]), hepatic metabolism of 14C-estradiol-17β (E2) at PND55 to PND62 was significantly higher in the 1000× compared to the control group, but hepatic detoxification enzyme activities had already returned to control values. The production of hepatic 2-hydroxy-E2 decreased, whereas that of estrone increased with age. In conclusion, the smallest dose of the mixture to induce significant effects was 100×, and mixture-induced changes in the hepatic metabolism of estrogens might be a sensitive indicator of persistent effects.

Effects of anesthetics and terminal procedures on biochemical and hormonal measurements in polychlorinated biphenyl treated rats.

This investigation reports the effects of various terminal procedures, and how they modified the responses to a toxicant (polychlorinated biphenyls [A1254], 130 mg/kg/day × 5 days) administered by gavage to Sprague-Dawley male rats. Terminal procedures included exsanguination via the abdominal aorta under anesthesia (isoflurane inhalation or Equithesin injection), decapitation with or without anesthesia, or narcosis induced by carbon dioxide inhalation. Effects of repeated anesthesia were also tested. Terminal procedures induced confounding stress responses, particularly when Equithesin was used. The terminal procedures modified the conclusions about effects of A1254 on the concentrations of corticosterone, insulin, glucagon, glucose, alkaline phosphatase, lactate dehydrogenase, uric acid, and blood urea nitrogen, from nonstatistically significant to significant changes, and in the case of luteinizing hormone from a statistically significant increase to a significant decrease. Investigations of effects of toxicants should be designed and interpreted considering potential changes induced by the selection of a terminal procedure.

Lack of effects of postnatal exposure to a mixture of aryl hydrocarbon-receptor agonists on the development of methylnitrosourea-induced mammary tumors in sprague-dawley rats.

There are concerns that early life exposure to organochlorines, including aryl hydrocarbon receptor (AhR) agonists, may lead to long-term effects and increase the risk of developing breast cancer. Our objective was to test if postnatal exposure to a mixture of 2,3,7,8-tetrachlorodibenzodioxin (TCDD)-like chemicals would modulate the development of methylnitrosourea (MNU)-induced mammary tumors. Females received by gavage a mixture containing 3 non-ortho-polychlorinated biphenyls (PCBs), 6 polychlorinated dibenzodioxins (PCDDs), and 7 polychlorinated dibenzofurans (PCDFs), at 1, 5, 10, 15, and 20d of age. The doses were equivalent to 0, 1, 10, 100, or 1000 times the amount ingested through breast milk by a human infant during its first 24 d of life. Subgroups of 1000× treated rats and controls were sacrificed at 21 d of age for assessment of mammary-gland development, cell death, and proliferation. Mammary-tumor development was assessed in MNU (30 mg/kg body weight ip at 50 days of age)-induced rats pre-exposed to the mixture (MNU-0, MNU-1, MNU-10, MNU-100, MNU-1000). Rats were sacrificed when their mammary tumors reached 1 cm in diameter, or when the rats reached ≥ 32 wk of age. Mammary-gland whole mounts were analyzed with all palpable and microscopic lesions (n=1563) histologically classified and grouped as benign, intraductal proliferations, or malignant. There were no marked effects on age at onset of puberty (vaginal opening) and estrous cyclicity. Despite a significant decrease in proliferating cell nuclear antigen (PCNA)-positive mammary cells in 1000× treated 21-d-old rats, there were no long-term dose-response effects on mammary-gland morphology and tumor development. In conclusion, postnatal exposure to the mixture of AhR agonists had no significant effects on the development of MNU-initiated mammary tumors.

Effects of lactational and/or in utero exposure to environmental contaminants on the glucocorticoid stress-response and DNA methylation of the glucocorticoid receptor promoter in male rats

Perinatal events can reprogram the hypothalamo-pituitary-adrenal axis for the entire lifespan leading to abnormal glucocorticoid stress-response (GSR) in adulthood: a phenomenon reported to be mediated by changes in DNA methylation of the glucocorticoid receptor (GR) gene promoter. We examined whether in utero and/or lactational exposure to mixtures of environmental contaminants can also induce abnormal GSR during adulthood. The experiment included nine treatment groups. From gestation day (GD) 0 until postnatal day (PND) 20, dams were fed daily with a cookie laced with corn oil (control) or a chemical mixture (M) [polychlorinated biphenyls (PCBs), organochlorine pesticides, and methylmercury] at 0.5 or 1.0mg/kg/day (0.5M, and M). At birth, some control (C) and M litters were cross-fostered to create four groups with the following in utero/postnatal exposure: C/C, M/C, C/M, M/M. Other dams received 1.8ng/kg/day of a mixture of aryl hydrocarbon receptor (AhR) agonists (non-ortho PCBs, PC-dibenzodioxins and PC-dibenzofurans) without or with 0.5M (0.5MAhR). In adult male offspring the abundance of GR in treated groups was not different from the control, but the AhR and M groups were significantly different from each other with opposite effects in the hippocampus and liver. There was no change in DNA methylation of the GR promoter (exon-17 and -110). Abnormal GSRs were detected in the AhR, 0.5MAhR, CM, and MM groups. The literature associates abnormal GSR with metabolic and mental health impairments, thus these results support further investigation of the influence of developmental exposure to environmental contaminants and predisposition to stress-induced diseases.

Sodium bisulfite pyrosequencing revealed that developmental exposure to environmental contaminant mixtures does not affect DNA methylation of DNA repeats in Sprague-Dawley rats

ABSTRACT Hypomethylation of DNA repeats has been linked to diseases and cancer predisposition. Human studies suggest that higher blood concentrations of environmental contaminants (EC) correlate with levels of hypomethylation of DNA repeats in blood. The objective of this study was to examine the effect of in utero and/or lactational exposure to EC on the methylation of DNA repeats (LINE-1 and identifier element) in Sprague-Dawley rat pups at birth, at postnatal day (PND) 21, and in adulthood (PND78–86). From gestation day 0 to PND20, dams were exposed to a mixture “M” of polychlorinated biphenyls (PCB), pesticides, and methylmercury (MeHg), at 0.5 or 1 mg/kg/d (0.5M and M). At birth, some control (C) and M litters were cross-fostered to create the following in utero/postnatal exposure groups: C/C, M/C, C/M, M/M. Additional dams received 1.8 ng/kg/d of a mixture of aryl-hydrocarbon receptor (AhR) agonists (non-ortho-PCB, PC-dibenzodioxins, and PC-dibenzofurans) without or with 0.5M (0.5MAhR). Measurements of EC residue levels confirmed differences in their accumulation across treatments, age, and tissues. Although induction of hepatic detoxification enzyme activities (cytochrome P-450) demonstrated biological effects of treatments, the assessment of methylation in DNA repeats by sodium bisulfite pyrosequencing of liver, spleen, and thymus samples revealed no marked treatment-related effects but significant tissue- and age-related methylation differences. Further studies are required to determine whether absence of significant observable treatment effects on methylation of DNA repeats in the rat relate to tissue, strain, or species differences.

Effects of Developmental Exposure to Mixtures of Environmental Contaminants on the Hepatic Metabolism of Estradiol-17β in Immature Female Sprague Dawley Rats

Exposure to environmental contaminants induces the activation of cytochrome P450s (CYP) which lead to the hydroxylation of contaminants and endogenous hormones such as estrogens. The hydroxylation of estrogens forms catecholestrogens (CEs), one of them being the mutagenic 4-hydroxyestradiol-17β (4−OH−E2). Catecholestrogens are transformed by catechol-o-methyltransferases (COMTs) into nonreactive methoxyestrogens. To investigate the hepatic metabolism of estradiol-17β in female offspring at postnatal day (PND) 21, pregnant rats were dosed daily from gestation day 1 until PND 21 with 2 dose levels of organochlorine pesticides (OCPs; 0.019 or 1.9 mg/kg per d), methylmercury (MeHg; 0.02 or 2 mg/kg per d), polychlorinated biphenyls (PCBs; 0.011 or 1.1 mg/kg per d), or a mixture (M; 0.05 or 5 mg/kg per d) including all 3 groups of chemicals. Concentrations of organochlorines in the mixture M were based on their proportions in serum of the Canadian Arctic population. The messenger RNA (mRNA) expressions of CYP and COMT were analyzed by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR). High-performance thin layer chromatography and phosphor imaging were used to measure the transformation of 14C substrates into estrogen metabolites. The low-dose treatments or the MeHg groups had no effect. The high-dose OCP, PCB, and M group increased the production of 2-OH-E2 and 6α-OH-E2, while only the PCB and M groups increased the 2-OH-CE/methoxyestrogen ratio. In all groups, the cytosolic COMT activity exceeded the microsomal production rate of 4-OH-E2. Although the M treatment included the PCB and OCP mixtures, it did not modify the estrogen metabolism more than did the PCB mixture alone. This endocrine disruption information contributes to our understanding of chemical interactions in the toxicology of chemical mixtures.