Diquat and paraquat are non-specific defoliants that induce toxicity in many organs including the lung, liver, kidney and brain. This toxicity is thought to be due to the generation of reactive oxygen species (ROS). An important pathway leading to ROS production by these compounds is redox cycling. In the present studies, diquat and paraquat redox cycling was characterized using human recombinant NADPH-cytochrome P450 reductase, rat liver microsomes, and Chinese Hamster Ovary (CHO) cells constructed to overexpress cytochrome P450 reductase (CHO-OR) and wild type control cells (CHO-WT). In redox cycling assays with recombinant cytochrome P450 reductase and microsomes, diquat was 10-40 times more effective in generating ROS when compared to paraquat (KM = 1.0 and 44.2 μM, respectively for H2O2 generation by diquat and paraquat using recombinant enzyme, and 15.1 and 178.5 μM, respectively for microsomes). In contrast, at saturating concentrations, these compounds showed similar redox cycling activity (Vmax ≈ 6.0 nmoles H2O2/min/mg protein) for recombinant enzyme and microsomes. Diquat and paraquat also redox cycle in CHO cells. Significantly more activity was evident in CHO-OR cells than CHO-WT cells. Diquat redox cycling in CHO cells was associated with marked increases in protein carbonyl formation, a marker of protein oxidation, as well as cellular oxygen consumption, measured using oxygen microsensors; greater activity was detected in CHO-OR cells than CHO-WT cells. These data demonstrate that ROS formation during diquat redox cycling can generate oxidative stress. Enhanced oxygen utilization during redox cycling may reduce intracellular oxygen available for metabolic reactions and contribute to toxicity.

Estrogens are major risk factors for the development of breast cancer; they can be metabolized to catechols, which are further oxidized to DNA-reactive quinones and semiquinones (SQs). These metabolites are mutagenic and may contribute to the carcinogenic activity of estrogens. Redox cycling of the SQs and subsequent generation of reactive oxygen species (ROS) is also an important mechanism leading to DNA damage. The SQs of exogenous estrogens have been shown to redox cycle, however, redox cycling and the generation of ROS by endogenous estrogens has never been characterized. In the present studies, we determined whether the catechol metabolites of endogenous estrogens, including 2-hydroxyestradiol, 4-hydroxyestradiol, 4-hydroxyestrone and 2-hydroxyestriol, can redox cycle in breast epithelial cells. These catechol estrogens, but not estradiol, estrone, estriol or 2-methoxyestradiol, were found to redox cycle and generate hydrogen peroxide (H(2)O(2)) and hydroxyl radicals in lysates of three different breast epithelial cell lines: MCF-7, MDA-MB-231 and MCF-10A. The generation of ROS required reduced nicotinamide adenine dinucleotide phosphate as a reducing equivalent and was inhibited by diphenyleneiodonium, a flavoenzyme inhibitor, indicating that redox cycling is mediated by flavin-containing oxidoreductases. Using extracellular microsensors, catechol estrogen metabolites stimulated the release of H(2)O(2) by adherent cells, indicating that redox cycling occurs in viable intact cells. Taken together, these data demonstrate that catechol metabolites of endogenous estrogens undergo redox cycling in breast epithelial cells, resulting in ROS production. Depending on the localized concentrations of catechol estrogens and enzymes that mediate redox cycling, this may be an important mechanism contributing to the development of breast cancer.

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Prochloraz is an imidazole fungicide, and its regulatory toxicological data package has been primarily generated in the 1990s. More recently, studies have been published demonstrating an interaction with hormone receptors/steroidogenesis and effects with an endocrine mode of action. In the present study, prochloraz has been investigated in a comprehensive in vivo study including relevant elements of current regulatory reproduction toxicity studies and additional mechanistic parameters. Prochloraz was administered per gavage in oil from GD 6 to PND 83 to pregnant and lactating Wistar rats and their respective offspring, at doses of 0.01 mg/kg bw/day (acceptable daily intake of prochloraz), 5 mg/kg bw/day [expected no-observed-effect-level (NOEL)] and 30 mg/kg bw/day. At 30 mg/kg bw/day maternal and offspring effects (decreased viability, lower number of live offspring) were seen including a delayed entry into male puberty (+1 day) accompanied by lower male offspring body weights, increased anogenital distance/index in females and transiently retained nipples in males at PND 12 (not seen at PND 20). The only finding at the "expected NOEL" was increased incidences of transiently retained nipples in males which are not considered adverse. No effects were seen in the low-dose group. There was no evidence for a non-monotonic dose-response curve or effects at low levels.

The current investigation examines whether the model anti-androgenic substance flutamide is capable of disrupting endocrine homeostasis at very low doses. The data generated clarify whether a non-monotonic dose-response relationship exists to enhance the current debate about the regulation of endocrine disruptors. Moreover, it is part of a series of investigations assessing the dose-response relationship of single and combined administration of anti-androgenic substances. A pre-postnatal in vivo study design was chosen, which was compliant with regulatory testing protocols. The test design was improved by additional endpoints addressing hormone levels, morphology, and histopathological examinations. Doses were chosen to represent a clear effect level (2.5 mg/kg bw/d), a low endocrine effect level (LOAEL, 0.25 mg/kg bw/d), a NOAEL for endocrine effects (0.025 mg/kg bw/d), a further dose at 0.0025 mg/kg bw/d flutamide, as well as an "ADI" (0.00025 mg/kg bw/d or 100-fold below the NOAEL) for the detection of a possible non-monotonic dose-response curve. Anti-androgenic changes were observable at LOAEL and the clear effect dose level but not at lower exposures. Nipple retention appeared to be the most sensitive measure of anti-androgenic effects, followed by age at sexual maturation, anogenital distance/anogenital index and male sex organ weights, as well as gross and histopathological findings. The results of all five doses indicate the absence of evidence for effects at very low dose levels. A non-monotonic dose-response relationship was not evident for the anti-androgenic drug flutamide.

Epoxiconazole (EPX; CAS-No. 133855-98-8) is a triazole class-active substance of plant protection products. At a dose level of 50 mg/kg bw/day, it causes a significantly increased incidence of late fetal mortality when administered to pregnant rats throughout gestation (gestation day [GD] 7-18 or 21), as reported previously (Taxvig et al., 2007, 2008) and confirmed in these studies. Late fetal resorptions occurred in the presence of significant maternal toxicity such as clear reduction of corrected body weight gain, signs of anemia, and, critically, a marked reduction of maternal estradiol plasma levels. Furthermore, estradiol supplementation at dose levels of 0.5 or 1.0 μg/animal/day of estradiol cyclopentylpropionate abolished the EPX-mediated late fetal resorptions. No increased incidences of external malformations were found in rats cotreated with 50 mg/kg bw/day EPX and estradiol cyclopentylpropionate, indicating that the occurrence of malformations was not masked by fetal mortality under the study conditions. Overall, the study data indicate that fetal mortality observed in rat studies with EPX is not the result of direct fetal toxicity but occurs indirectly via depletion of maternal estradiol levels. The clarification of the human relevance of the estrogen-related mechanism behind EPX-mediated late fetal resorptions in rats warrants further studies. In particular, this should involve investigation of the placenta (Rey Moreno et al., 2013), since it is the materno-fetal interface and crucial for fetal maintenance. The human relevance is best addressed in a species which is closer to humans with reference to placentation and hormonal regulation of pregnancy, such as the guinea pig (Schneider et al., 2013).

Tox21 and ToxCast are high-throughput in vitro screening programs coordinated by the U.S. National Toxicology Program and the U.S. Environmental Protection Agency, respectively, with the goal of forecasting biological effects in vivo based on bioactivity profiling. The present study investigated whether mechanistic insights in the biological targets of food-relevant chemicals can be obtained from ToxCast results when the chemicals are grouped according to structural similarity. Starting from the 556 direct additives that have been identified in the ToxCast database by Karmaus et al. [Karmaus, A. L., Trautman, T. D., Krishan, M., Filer, D. L., and Fix, L. A. (2017). Curation of food-relevant chemicals in ToxCast. Food Chem. Toxicol. 103, 174–182.], the results showed that, despite the limited number of assays in which the chemical groups have been tested, sufficient results are available within so-called “DNA binding” and “nuclear receptor” target families to profile the biological activities of the defined chemical groups for these targets. The most obvious activity identified was the estrogen receptor-mediated actions of the chemical group containing parabens and structurally related gallates, as well the chemical group containing genistein and daidzein (the latter 2 being particularly active toward estrogen receptor β as a potential health benefit). These group effects, as well as the biological activities of other chemical groups, were evaluated in a series of case studies. Overall, the results of the present study suggest that high-throughput screening data could add to the evidence considered for regulatory risk assessment of food chemicals and to the evaluation of desirable effects of nutrients and phytonutrients. The data will be particularly useful for providing mechanistic information and to fill data gaps with read-across.

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