In vivo assessment of respiratory burst inhibition by xenobiotic exposure using larval zebrafish.

Currently, assessment of the potential immunotoxicity of a given agent involves a tiered approach for hazard identification and mechanistic studies, including observational studies, evaluation of immune function, and measurement of susceptibility to infectious and neoplastic diseases. These studies generally use costly low-throughput mammalian models. Zebrafish, however, offer an excellent alternative due to their rapid development, ease of maintenance, and homology to mammalian immune system function and development. Larval zebrafish also are a convenient model to study the innate immune system with no interference from the adaptive immune system. In this study, a respiratory burst assay (RBA) was utilized to measure reactive oxygen species (ROS) production after developmental xenobiotic exposure. Embryos were exposed to non-teratogenic doses of chemicals and at 96 h post-fertilization, the ability to produce ROS was measured. Using the RBA, 12 compounds with varying immune-suppressive properties were screened. Seven compounds neither suppressed nor enhanced the respiratory burst; five reproducibly suppressed global ROS production, but with varying potencies: benzo[a]pyrene, 17ß-estradiol, lead acetate, methoxychlor, and phenanthrene. These five compounds have all previously been reported as immunosuppressive in mammalian innate immunity assays. To evaluate whether the suppression of ROS by these compounds was a result of decreased immune cell numbers, flow cytometry with transgenic zebrafish larvae was used to count the numbers of neutrophils and macrophages after chemical exposure. With this assay, benzo[a]pyrene was found to be the only chemical that induced a change in the number of immune cells by increasing macrophage but not neutrophil numbers. Taken together, this work demonstrates the utility of zebrafish larvae as a vertebrate model for identifying compounds that impact innate immune function at non-teratogenic levels and validates measuring ROS production and phagocyte numbers as metrics for monitoring how xenobiotic exposure alters the innate immune system.

Methoxychlor exposure induces oxidative stress and affects mouse oocyte meiotic maturation.

Methoxychlor (MXC) is used worldwide against insects and other pests. This organochlorine pesticide acts as a xenoestrogen, promotes oxidative stress, and is considered cytotoxic and genotoxic, causing abortions and stillbirths in females. Mechanistically related estrogens and oxidants affect oocyte meiosis, so we investigated the effects of MXC on mouse oocyte meiotic maturation. Our results showed that maturation rates of MXC-treated oocytes were lower than those of controls, which was due to abnormal spindle morphologies and DNA double-strand breaks, as confirmed by increased γ-H2AX foci. Our findings also suggest that MXC may affect oocyte quality by causing the accumulation of superoxide radicals and other reactive oxygen species, aberrant mitochondrial distribution, decreased mitochondrial membrane potential, and increased lipid peroxidation. Thus, exposure to MXC negatively affects oocyte meiotic maturation, primarily through impairments in cellular ROS metabolism. Mol. Reprod. Dev. 83: 768-779, 2016 © 2016 Wiley Periodicals, Inc.

Prior exposure to immunosuppressive organophosphorus or organochlorine compounds aggravates the T(H)1- and T(H)2-type allergy caused by topical sensitization to 2,4-dinitrochlorobenzene and trimellitic anhydride.

Immunosuppressive environmental chemicals may increase the potency of allergens and thereby play a role in the development of allergic diseases. This study's primary objective was to examine the mechanisms behind the relationship between allergic diseases and the immunosuppression induced by some environmental chemicals. We focused on the modulation of allergic potential in vitro and in mice by the organophosphorus pesticide O,O-diethyl-O-4-nitrophenyl-thiophosphate (parathion) and the organochlorine pesticide 1,1,1-trichloro-2,2-bis(4-methoxy-phenyl)ethane (methoxychlor), with respect to the T(H)1-type allergen 2,4-dinitrochlorobenzene (DNCB) and the T(H)2-type allergen trimellitic anhydride (TMA). Mice (4-week-old) were orally administered parathion or methoxychlor. Four weeks after the final dosing, the mice were sensitized to DNCB or TMA, and T-lymphocyte proliferation measured in their (using a local lymph node assay [LLNA]). In addition, we analyzed T-lymphocytes via surface antigen expression and local cytokine production in auricular lymph nodes after treatment with 0.1% DNCB or 0.3% TMA. The estimated concentration of DNCB and TMA to yield a stimulation index (SI) of cell proliferation of three decreased markedly in parathion- and methoxychlor-pre-treated mice. Pesticide pre-treatment induced marked increases in the number of helper and cytotoxic T-cells, levels of T(H)1 and T(H)2 cytokines, and gene expression in lymph node cells. According to our results, T(H)1- and T(H)2-type allergies are aggravated by prior exposure to immunosuppressive environmental chemicals.

Estrogenic endocrine disruptive components interfere with calcium handling and differentiation of human trophoblast cells.

During development, calcium (Ca) is actively transported by placental trophoblasts to meet fetal nutritional and the skeletal mineralization needs. Maternal exposure to estrogenic pesticides, such as 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and methoxychlor (MTC), has been shown to result in reproductive disorders and/or abnormal fetal development. In this study, we have examined the effects of exposure of trophoblastic cells to MTC and DTT, in comparison to 17beta-estradiol (E2) and diethylstilbestrol (DES), to test the hypothesis that cellular Ca handling is a target for these endocrine disruptive components. Treatment with DDT, MTC, DES, or E2 increased cellular Ca uptake, and the expression of trophoblast-specific human Ca binding protein (HCaBP) was down-regulated by both MTC and DDT. Treatment with MTC, DDT, and DES inhibited cell proliferation, induced apoptosis, and suppressed expression of several trophoblast differentiation marker genes. These effects were reversed by overexpression of metallothionein IIa, a gene highly responsive to cadmium and other metals. These results strongly suggest that trophoblast Ca handling functions are endocrinally modulated, and that their alteration by candidate endocrine disruptors, such as MTC and DDT, constitutes a possible pathway of the harmful effects of these components on fetal development.

[Chlorinated hydrocarbon insecticides(DDT, methoxychlor, HCH etc.)].

Endocrine disrupting properties of organochlorine(chlorinated hydrocarbon) insecticides are reviewed. Physicochemical properties such as water solubility and partition coefficient of the organochlorine insecticides greatly influence their environmental fates. Insolubility in water and high partition coefficient usually enhance the environmental persistence of the compounds. On the endocrine disrupting properties, an important metabolite of p,p'-DDT, p,p'-DDE, is known to exhibit weak anti-androgenic properties. An isomer o,p'-DDT is estrogenic. Metabolites of methoxychlor, mono-demethylated and di-demethylated methoxychlor derivatives, are weakly estrogenic. Their dehydochlorinated product also exhibits estrogenicity. HCH isomers are environmentally persistent compounds, but there is few reports on their endocrine disrupting properties. Some other organochlorine pesticides such as cyclodienes and chlordecone is reported to be estrogenic.

Low-dose bioactivity of xenoestrogens in animals: fetal exposure to low doses of methoxychlor and other xenoestrogens increases adult prostate size in mice.

The hormonal activity of natural estrogens is influenced by the degree to which they bind to serum proteins. In the pregnant female and in the fetus, greater than 99% of estradiol may be bound by serum binding proteins. Therefore, even though total serum levels of estradiol appear very high in fetuses, we have found that in rodent fetuses, there is a very low free concentration of estradiol (0.2 pg/ml). Naturally occurring variation in fetal serum estradiol predicts differences in numerous postnatal traits, including prostate size. In addition, when this low level of free estradiol was experimentally increased from 0.2 to 0.3 pg/ml during the last third of fetal life, treated male mice showed an increase in adult prostate weight. Fetal exposure to low doses of xenobiotic estrogens by feeding to pregnant females, including the compounds methoxychlor (20 and 2000 micrograms/kg body weight), DES (0.02 to 2 micrograms/kg body weight) and bisphenol A (2 and 20 micrograms/kg body weight), also led to increased prostate weight in adulthood. In contrast, fetal doses of natural estradiol and DES above the physiological range of estrogenic activity, and within a toxicological dose range, led to the opposite outcome, a reduction in subsequent adult prostate weight. This indicates that it may be impossible to assess endocrine-disrupting activities in response to low doses within a physiological range of activity by using high, toxic doses of xenoestrogens in testing procedures. We have developed approaches in vitro to predict the potential estrogenic bioactivity of compounds in the physiologically relevant range in animals and humans. We address the following factors in predicting the final observed endocrine-disrupting effect in the animal: (1) the intrinsic estrogenic activity of a given molecule, (2) the effective free concentration determined by how the molecule is carried in serum, (3) partitioning between aqueous and lipid compartments in body and cell lipids, and (4) absorption and metabolism relative to the route of exposure. The studies and strategies we describe are important in developing criteria for a tiered testing system for the detection of estrogenic chemicals as well as endocrine-disrupting chemicals with different modes of action.