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Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are used in personal and medical care products. In the ovary, antral follicles are essential for steroidogenesis and ovulation. DBP, BBP, and DEHP are known to inhibit mouse antral follicle growth and ovulation in vitro, and associate with decreased antral follicle counts in women. Given that the in vivo effects of a three-phthalate mixture on antral follicles are unknown, we evaluated the effects of a human-relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. Adult CD-1 female mice were fed corn oil (vehicle), or two dose levels of a phthalate mixture based on estimated exposures in general (32 µg/kg/d; PHT 32) and occupationally exposed (500 µg/kg/d; PHT 500) populations for 10 d. Antral follicles (>250 µm) were isolated and subjected to proteome profiling via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were detected, of which 194 were differentially abundant between vehicle and PHT 32, and 136 between vehicle and PHT 500. Bioinformatic analysis revealed significantly different responses between the two phthalate doses. Protein abundance differences in the PHT 32 exposure mapped to cytoplasm, mitochondria, and lipid metabolism; whereas those in the PHT 500 exposure mapped to cytoplasm, nucleus, and phosphorylation. When both doses altered proteins mapped to common processes, the associated predicted transcription factors were different. These findings provide novel mechanistic insight into phthalate-associated, ovary-driven reproductive outcomes in women., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phthalates are compounds used in consumer and medical products worldwide. Phthalate exposure in women has been demonstrated by detection of phthalate metabolites in their urine and ovarian follicular fluid. High urinary phthalate burden has been associated with reduced ovarian reserve and oocyte retrieval in women undergoing assisted reproduction. Unfortunately, no mechanistic explanation for these associations is available. In short term in vivo and in vitro animal studies modeling human-relevant exposures to di-n-butyl phthalate (DBP), we have identified ovarian folliculogenesis as a target for phthalate exposures. In the present study, we investigated whether DBP exposure negatively influences insulin-like growth factor 1 (IGF1) signaling in the ovary and disrupts ovarian folliculogenesis. CD-1 female mice were exposed to corn oil (vehicle) or DBP (10 µg/kg/day, 100 µg/kg/day, or 1000 mg/kg/day) for 20-32 days. Ovaries were collected as animals reached the proestrus stage to achieve estrous cycle synchronization. Levels of mRNAs encoding IGF1 and 2 (Igf1 and Igf2), IGF1 receptor (Igf1r), and IGF-binding proteins 1-6 (Ifgbp1-6) were measured in whole ovary homogenates. Ovarian follicle counts and immunostaining for phosphorylated IGF1R protein (pIGF1R) were used to evaluate folliculogenesis and IGF1R activation, respectively. DBP exposure, at a realistic dose that some women may experience (100 µg/kg/day for 20-32 days), reduced ovarian Igf1 and Igf1r mRNA expression and reduced small ovarian follicle numbers and primary follicle pIGF1R positivity in DBP-treated mice. These findings reveal that DBP tampers with the ovarian IGF1 system and provide molecular insight into how phthalates could influence the ovarian reserve in females., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Excretion of toxicants accumulated from firefighter exposures through breastmilk represents a potential hazard. We investigated if firefighting exposures could increase the concentration of polybrominated diphenyl ethers (PBDEs) and aryl hydrocarbon receptor (AhR) activation in excreted breastmilk. Firefighters and non-firefighters collected breastmilk samples prior to any firefighting responses (baseline) and at 2, 8, 24, 48, and 72 hours after a structural fire (firefighters only). Five PBDE analytes (BDEs 15, 28, 47, 99, and 153) detected in at least 90% of samples were summed for analyses. The AhR in vitro DR CALUX® bioassay assessed the mixture of dioxin-like compounds and toxicity from breastmilk extracts. Baseline PBDEs and AhR response were compared between firefighters and non-firefighters. Separate linear mixed models assessed changes in sum of PBDEs and AhR response among firefighters over time and effect modification by interior or exterior response was assessed. Baseline PBDE concentrations and AhR responses did not differ between the 21 firefighters and 10 non-firefighters. There were no significant changes in sum of PBDEs or AhR response among firefighters over time post-fire, and no variation by interior or exterior response. Plots of sum of PBDEs and AhR response over time demonstrated individual variation but no consistent pattern. Currently, our novel study results do not support forgoing breastfeeding after a fire exposure. However, given study limitations and the potential hazard of accumulated toxicants from firefighter exposures excreted via breastfeeding, future studies should consider additional contaminants and measures of toxicity by which firefighting may impact maternal and child health., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology.)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the classic aryl hydrocarbon receptor (AhR) agonist, is a potent environmental toxicant and endocrine-disrupting chemical (EDC) with known developmental toxicity in humans, rodents, and fish. Early life exposure to some EDCs, including TCDD, is linked to the occurrence of adult-onset and multigenerational disease. Previous work exposing juvenile F0 zebrafish (Danio rerio) to 50 ppt (parts per trillion) TCDD during reproductive development has shown male-mediated transgenerational decreases in fertility (F0-F2) and histologic and transcriptomic alterations in F0 testes. Here, we analyzed male germline alterations in F1 and F2 adult fish, looking for changes in testicular histology and gene expression inherited through the male lineage that could account for decreased reproductive capacity. Testes of TCDD-lineage F1 fish displayed an increase in spermatogonia (immature germ cells) and decrease in spermatozoa (mature germ cells). No histological changes were present in F2 fish. Transcriptomic analysis of exposed F1 and F2 testes revealed alterations in lipid and glucose metabolism, oxidation, xenobiotic response, and sperm cell development and maintenance genes, all of which are implicated in fertility outcomes. Overall, we found that differential expression of reproductive genes and reduced capacity of sperm cells to mature could account for the reproductive defects previously seen in TCDD-exposed male zebrafish and their descendants, providing insight into the distinct multigenerational effects of toxicant exposure.

Flavonoids are a large, structurally diverse class of bioactive naturally occurring chemicals commonly detected in breast milk, soy based infant formulas, amniotic fluid, and fetal cord blood. The potential for pervasive early life stage exposures raises concerns for perturbation of embryogenesis, though developmental toxicity and bioactivity information is limited for many flavonoids. Therefore, we evaluated a suite of 24 flavonoid and flavonoid-like chemicals using a zebrafish embryo-larval toxicity bioassay-an alternative model for investigating developmental toxicity of environmentally relevant chemicals. Embryos were exposed to 1-50 µM of each chemical from 6 to 120 h postfertilization (hpf), and assessed for 26 adverse developmental endpoints at 24, 72, and 120 hpf. Behavioral changes were evaluated in morphologically normal animals at 24 and 72 hpf, at 120 hpf using a larval photomotor response (LPR) assay. Gene expression was comparatively evaluated for all compounds for effects on biomarker transcripts indicative of AHR (cyp1a) and ER (cyp19a1b, esr1, lhb, vtg) pathway bioactivity. Overall, 15 of 24 flavonoids elicited adverse effects on one or more of the developmental or behavioral endpoints. Hierarchical clustering and principle component analyses compared toxicity profiles and identified 3 distinct groups of bioactive flavonoids. Despite robust induction of multiple estrogen-responsive biomarkers, co-exposure with ER and GPER antagonists did not ameliorate toxicity, suggesting ER-independence and alternative modes of action. Taken together, these studies demonstrate that development is sensitive to perturbation by bioactive flavonoids in zebrafish that are not related to traditional estrogen receptor mode of action pathways. This integrative zebrafish platform provides a useful framework for evaluating flavonoid developmental toxicity and hazard prioritization., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

TGFβ2 (transforming growth factor-β2) is a key growth factor regulating epithelial to mesenchymal transition (EMT). TGFβ2 triggers cardiac progenitor cells to differentiate into mesenchymal cells and give rise to the cellular components of coronary vessels as well as cells of aortic and pulmonary valves. TGFβ signaling is dependent on a dynamic on and off switch in Smad activity. Arsenite exposure of 1.34 μM for 24-48 h has been reported to disrupt Smad phosphorylation leading to deficits in TGFβ2-mediated cardiac precursor differentiation and transformation. In this study, the molecular mechanism of acute arsenite toxicity on TGFβ2-induced Smad2/3 nuclear shuttling and TGFβ2-mediated cardiac EMT was investigated. A 4-h exposure to 5 μM arsenite blocks nuclear accumulation of Smad2/3 in response to TGFβ2 without disrupting Smad phosphorylation or nuclear importation. The depletion of nuclear Smad is restored by knocking-down Smad-specific exportins, suggesting that arsenite augments Smad2/3 nuclear exportation. The blockage in TGFβ2-Smad signaling is likely due to the loss of Zn(2+) cofactor in Smad proteins, as Zn(2+) supplementation reverses the disruption in Smad2/3 nuclear translocation and transcriptional activity by arsenite. This coincides with Zn(2+) supplementation rescuing arsenite-mediated deficits in cardiac EMT. Thus, zinc partially protects cardiac EMT from developmental toxicity by arsenite., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Polycyclic aromatic hydrocarbons (PAHs) are priority environmental contaminants that exhibit mutagenic, carcinogenic, proinflammatory, and teratogenic properties. Oxygen-substituted PAHs (OPAHs) are formed during combustion processes and via phototoxidation and biological degradation of parent (unsubstituted) PAHs. Despite their prevalence both in contaminated industrial sites and in urban air, OPAH mechanisms of action in biological systems are relatively understudied. Like parent PAHs, OPAHs exert structure-dependent mutagenic activities and activation of the aryl hydrocarbon receptor (AHR) and cytochrome p450 metabolic pathway. Four-ring OPAHs 1,9-benz-10-anthrone (BEZO) and benz(a)anthracene-7,12-dione (7,12-B[a]AQ) cause morphological aberrations and induce markers of oxidative stress in developing zebrafish with similar potency, but only 7,12-B[a]AQ induces robust Cyp1a protein expression. We investigated the role of the AHR in mediating the toxicity of BEZO and 7,12-B[a]AQ, and found that knockdown of AHR2 rescued developmental effects caused by both compounds. Using RNA-seq and molecular docking, we identified transcriptional responses that precede developmental toxicity induced via differential interaction with AHR2. Redox-homeostasis genes were affected similarly by these OPAHs, while 7,12-B[a]AQ preferentially activated phase 1 metabolism and BEZO uniquely decreased visual system genes. Analysis of biological functions and upstream regulators suggests that BEZO is a weak AHR agonist, but interacts with other transcriptional regulators to cause developmental toxicity in an AHR-dependent manner. Identifying ligand-dependent AHR interactions and signaling pathways is essential for understanding toxicity of this class of environmentally relevant compounds., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The increased use of flammable plastics and electronic devices along with stricter fire safety standards has led to the heavy use of flame retardant chemicals in many consumer, commercial, and industrial products. Although flame retardant use has increased, a great deal of uncertainty surrounds their safety with some evidence showing toxicity and risk to human and environmental health. Recent efforts have focused on designing high-throughput biological platforms with nonmammalian models to evaluate and prioritize chemicals with limited hazard information. To complement these efforts, this study used a new morphological and behavioral testing platform with embryonic zebrafish to characterize the developmental toxicity of 44 halogenated and organophosphate flame retardants, including several of their known metabolites. Zebrafish were exposed to flame retardants from 6 to 120 h post fertilization (hpf) across concentrations spanning 4 orders of magnitude (eg, 6.4 nM to 64 µM). Flame retardant effects on survival and development were evaluated at 24 and 120 hpf, and neurobehavioral changes were measured using 2 photomotor response (PMR) assays. Compared to controls, 93% (41/44) of flame retardants studied elicited adverse effects among one or more of the bioassays and concentrations tested with the aryl phosphate ester (APE)-based mono-isopropylated triaryl phosphate and the brominated-bisphenol-A analog tetrabromobisphenol-A producing the greatest array of malformations. Hierarchical clustering showed that APE flame retardants with isopropyl, butyl, and cresyl substituents on phenyl rings clustered tightly and were particularly potent. Both PMR assays were highly predictive of morphological defects supporting their use as nonlethal means of evaluating teratogenicity that could allow for additional evaluations of long-term or delayed effects in older animals. Taken together, evidence presented here indicates that zebrafish neurodevelopment is highly sensitive to many flame retardants currently in use and can be used to understand potential vulnerabilities to human health., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Arsenic exposure during embryonic development can cause ischemic heart pathologies later in adulthood which may originate from impairment in proper blood vessel formation. The arsenic-associated detrimental effects are mediated by arsenite (iAs(III)) and its most toxic metabolite, monomethylarsonous acid [MMA (III)]. The impact of MMA (III) on coronary artery development has not yet been studied. The key cellular process that regulates coronary vessel development is the epithelial-mesenchymal transition (EMT). During cardiac EMT, activated epicardial progenitor cells transform to mesenchymal cells to form the cellular components of coronary vessels. Smad2/3 mediated TGFβ2 signaling, the key regulator of cardiac EMT, is disrupted by arsenite exposure. In this study, we compared the cardiac toxicity of MMA (III) with arsenite. Epicardial progenitor cells are 15 times more sensitive to MMA (III) cytotoxicity when compared with arsenite. MMA (III) caused a significant blockage in epicardial cellular transformation and invasion at doses 10 times lower than arsenite. Key EMT genes including TGFβ ligands, TβRIII, Has2, CD44, Snail1, TBX18, and MMP2 were down regulated by MMA (III) exposure. MMA (III) disrupted Smad2/3 activation at a dose 20 times lower than arsenite. Both arsenite and MMA (III) significantly inhibited Erk1/2 and Erk5 phosphorylation. Nuclear translocation of Smad2/3 and Erk5 was also blocked by arsenical exposure. However, p38 activation, as well as smooth muscle differentiation, was refractory to the inhibition by the arsenicals. Collectively, these findings revealed that MMA (III) is a selective disruptor of cardiac EMT and as such may predispose to arsenic-associated cardiovascular disorders., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Dioxin is a ubiquitous environmental pollutant that induces toxicity when bound to the aryl hydrocarbon receptor (AhR). Significant differences in susceptibility of mouse strains to dioxin toxicity are largely accounted for by the dissociation constant of binding to dioxins of AhR subtypes encoded by different alleles. We showed that cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1), components of a prostanoid synthesis pathway, play essential roles in the onset of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced hydronephrosis of neonatal mice. Although C57BL/6J and BALB/cA mice harbor AhR receptors highly responsive to TCDD, they were found by chance to differ significantly in the incidence of TCDD-induced hydronephrosis. Therefore, the goal of the present study was to determine the molecular basis of this difference in susceptibility to TCDD toxicity. For this purpose, we administered C57BL/6J and BALB/cA dams' TCDD at an oral dose of 15 or 80 μg/kg on postnatal day (PND) 1 to expose pups to TCDD via lactation, and the pups' kidneys were collected on PND 7. The incidence of hydronephrosis in C57BL/6J pups (64%) was greater than in BALB/cA pups (0%, p < 0.05), despite similarly increased levels of COX-2 mRNA. The incidence of hydronephrosis in these mouse strains paralleled the levels of renal mPGES-1 mRNA and early growth response 1 (Egr-1) that modulates mPGES-1 gene expression, as well as PGE2 concentrations in urine. Although these mouse strains possess AhR alleles tightly bound to TCDD, their difference in incidence and severity of hydronephrosis can be explained, in part, by differences in the expression of mPGES-1 and Egr-1., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

There are tens of thousands of man-made chemicals in the environment; the inherent safety of most of these chemicals is not known. Relevant biological platforms and new computational tools are needed to prioritize testing of chemicals with limited human health hazard information. We describe an experimental design for high-throughput characterization of multidimensional in vivo effects with the power to evaluate trends relating to commonly cited chemical predictors. We evaluated all 1060 unique U.S. EPA ToxCast phase 1 and 2 compounds using the embryonic zebrafish and found that 487 induced significant adverse biological responses. The utilization of 18 simultaneously measured endpoints means that the entire system serves as a robust biological sensor for chemical hazard. The experimental design enabled us to describe global patterns of variation across tested compounds, evaluate the concordance of the available in vitro and in vivo phase 1 data with this study, highlight specific mechanisms/value-added/novel biology related to notochord development, and demonstrate that the developmental zebrafish detects adverse responses that would be missed by less comprehensive testing strategies.

Hydronephrosis induced in the kidney of neonatal mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via lactation is a sensitive and characteristic hallmark of TCDD teratogenicity. We previously found that cyclooxygenase-2 (COX-2) activity induced in mouse neonate kidneys by lactational TCDD exposure is required for this toxicity. COX-2 is an inducible form of cyclooxygenase and is responsible for producing prostaglandins (PGs) and thromboxane. PGE(2), a prostaglandin, is elevated in TCDD-exposed mouse pups. In this study, we investigated the role of microsomal prostaglandin E synthase-1 (mPGES-1), an inducible form of PGE(2) synthase, in TCDD-induced hydronephrosis. A dose of 10 μg TCDD/kg to dams increased mPGES-1 messenger RNA abundance, urinary PGE(2) levels, and the incidence of hydronephrosis in mPGES-1 wild-type pups. In homozygous mPGES-1 knockout (KO) mice, in contrast, TCDD-induced hydronephrosis was suppressed, demonstrating an essential role of mPGES-1 in the response. Lack of the mPGES-1 gene also suppressed urinary PGE(2) level to near the basal level in TCDD-exposed pups. In conclusion, mPGES-1 upregulation upon lactational TCDD exposure is a causal factor for TCDD-induced hydronephrosis in mouse neonates.

The mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase signaling cascades have been implicated in a number of human cancers. The tumor suppressor gene tuberous sclerosis-2 (Tsc-2) functions as a negative regulator of mTOR. Critical proteins in both pathways are activated following treatment of Eker rats (Tsc-2(EK/+)) with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ), which also results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. Western blot analysis of kidney tumors formed following treatment of Tsc-2(EK/+) rats with TGHQ for 8 months revealed increases in B-Raf, Raf-1, pERK, cyclin D1, 4EBP1, and p-4EBP1-Ser65, -Thr70, and -Thr37/46 expression. Similar changes are observed following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (quinol-thioether rat renal epithelial [QTRRE] cells) that are also null for tuberin. These cells exhibit high ERK, B-Raf, and Raf-1 kinase activity and increased expression of all p-4EBP1s and cyclin D1. Treatment of the QTRRE cells with the Raf kinase inhibitor, sorafenib, or the MEK1/2 kinase inhibitor, PD 98059, produced a significant decrease in the protein expression of all p-4EBP1s and cyclin D1. Following siRNA knockdown of Raf-1, Western blot analysis revealed a significant decrease in Raf-1, cyclin D1, and all p-4EBP1 forms noted above. In contrast, siRNA knockdown of B-Raf resulted in a nominal change in these proteins. The data indicate that Raf-1/MEK/ERK participates in crosstalk with 4EBP1, which represents a novel pathway interaction leading to increased protein synthesis, cell growth, and kidney tumor formation.

MicroRNA (miRNA) is a class of small RNA that functions as a negative regulator of gene expression. Human and mouse genomes encode over 1400 and 700 miRNAs, respectively, and most of the cellular pathways are considered to be modulated by miRNAs. However, the pathophysiological role of miRNAs is still largely unknown. Thus, we investigated the possible involvement of miRNAs in the toxic responses to xenobiotic chemicals. Here, we searched for miRNAs responsible for inducing liver damage in mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and found that miR-101a and miR-122 are differentially downregulated by TCDD in a time-dependent manner. Because miRNA exerts multiple actions by repressing its target genes, we quantified the target genes of miR-101a, such as cyclooxygenase-2 (COX-2), enhancer of zeste homolog 2, and cFos, and found the upregulation of these genes, which suggests that miR-101a downregulates the expressions of these genes in the mouse liver. A COX-2 selective inhibitor, NS-398, suppressed the onset of TCDD-induced liver damage. In conclusion, this study demonstrated that TCDD dysregulates the expression of miR101a and miR122 and that COX-2, a target gene of miR101a, plays a significant role in liver damage in mice exposed to TCDD.

The loss of tuberin, the tuberous sclerosis-2 (Tsc-2) gene product, is associated with cytoplasmic mislocalization of p27 in uterine leiomyomas derived from Eker rats (Tsc-2(EK/+)) and in human metastatic renal cell carcinoma tissue. Signaling associated with cytoplasmic mislocalization of p27 in renal cancer is relatively unknown. Renal tumors derived from 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-treated Tsc-2(EK/+) rats, and null for tuberin, display elevated nuclear and cytosolic p27, with parallel increases in cytosolic cyclin D1 levels. Similar changes are observed in TGHQ-transformed renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), which, in addition to the cytoplasmic mislocalization of p27 and cyclin D1, exhibit high ERK, B-Raf, and Raf-1 kinase activity. Renal tumor xenografts, derived from subcutaneous injection of QTRRE cells into nude mice, also display increases in cytosolic mislocalization of p27 and cyclin D1. Dibutyryl cAMP and/or phosphodiesterase inhibitors (PIs; pentoxifylline or theophylline) increase Rap1B activation, B-Raf kinase activity, and cytosolic p27/cyclin D1 protein levels in QTRRE cells. Inhibition of Raf kinases with either sorafenib or B-Raf small interfering RNA (siRNA) caused a mitogen-activated protein kinase-mediated downregulation of p27. Moreover, decreases in cyclin D1 were also associated with p27 siRNA knockdown in QTRRE cells. Finally, theophylline-mediated increases in p27 and cyclin D1 were attenuated by sorafenib, which modulated Raf/MEK/ERK signaling. Collectively, these data suggest that the cAMP/Rap1B/B-Raf pathway modulates the expression of p27 and the cytoplasmic mislocalization of p27-cyclin D1 in tuberous sclerosis gene-regulated-renal cancer. Therefore, the loss of tuberin and engagement of the cAMP pathway may independently direct p27-cyclin D1 cytosolic stabilization during renal tumor formation.

Electrophile-mediated post-translational modifications (PTMs) are known to cause tissue toxicities and disease progression. These effects are mediated via site-specific modifications and structural disruptions associated with such modifications. 1,4-Benzoquinone (BQ) and its quinone-thioether metabolites are electrophiles that elicit their toxicity via protein arylation and the generation of reactive oxygen species. Site-specific BQ-lysine adducts are found on residues in cytochrome c that are necessary for protein-protein interactions, and these adducts contribute to interferences in its ability to facilitate apoptosome formation. To further characterize the structural and functional impact of these BQ-mediated PTMs, the original mixture of BQ-adducted cytochrome c was fractionated by liquid isoelectric focusing to provide various fractions of BQ-adducted cytochrome c species devoid of the native protein. The fractionation process separates samples based on their isoelectric point (pI), and because BQ adducts form predominantly on lysine residues, increased numbers of BQ adducts on cytochrome c correlate with a lower protein pI. Each fraction was analyzed for structural changes, and each was also assayed for the ability to support apoptosome-mediated activation of caspase-3. Circular dichroism revealed that several of the BQ-adducted cytochrome c species maintained a slightly more rigid structure in comparison to native cytochrome c. BQ-adducted cytochrome c also failed to activate caspase-3, with increasing numbers of BQ-lysine adducts corresponding to a greater inability to activate the apoptosome. In summary, the specific site of the BQ-lysine adducts, and the nature of the adduct, are important determinants of the subsequent structural changes to cytochrome c. In particular, adducts at sites necessary for protein-protein interactions interfere with the proapoptotic function of cytochrome c.

"All models are wrong, some models are useful." George E.P. Box, "Robustness in the strategy of scientific model building", 1979. This quote from George Box was the title of a book chapter on mathematical models-exactly the type of models which we will not address here, although a lot of the general reasoning can as well be translated to them, especially because they depend on the input and thus on the limitations of either in vitro or in vivo data, when applied for regulatory toxicology. This article was prompted by the 2008 SOT/EuroTox debate (March 2008 in Seattle and October 2008 in Rhodes), which challenged us in an Hegelian approach as thesis and antithesis to present with changing roles on both occasions on the statement "In vitro tests are useless for regulatory use." Here, we would like following Hegel a summary of thesis and antithesis, but also try to outline the synthesis.

Adult zebrafish can completely regenerate their caudal fin following amputation. This complex process is initiated by the formation of an epithelial wound cap over the amputation site by 12 h post amputation (hpa). Once the cap is formed, mesenchymal cells proliferate and migrate from sites distal to the wound plane and accumulate under the epithelial cap forming the blastemal structure within 48 hpa. Blastemal cells proliferate and differentiate, replacing the amputated tissues, which are populated with angiogenic vessels and innervating nerves during the regenerative outgrowth phase which is completed around 14 days post amputation (dpa). Regenerative outgrowth does not occur in TCDD-exposed zebrafish. To identify the molecular pathways that are perturbed by TCDD exposure, male zebrafish were ip injected with 50 ng/g TCDD or vehicle and caudal fins were amputated. Regenerating fin tissue was collected at 1, 3, and 5 dpa for mRNA abundance analysis. Microarray analysis and quantitative real time PCR revealed that wound healing and regeneration alone altered the expression of nearly 900 genes by at least two-fold between 1 and 5 dpa. TCDD altered the abundance of 370 genes at least two-fold. Among these, several known aryl hydrocarbon responsive genes were identified in addition to several genes involved in extracellular matrix composition and metabolism. The profile of misexpressed genes is suggestive of impaired cellular differentiation and extracellular matrix composition potentially regulated by Sox9b.

We have previously shown that exposure of mice to 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) induces activation-like changes in splenic dendritic cells (DC) in the absence of antigen challenge. Since activation of DC reduces their ability to phagocytize antigen, we examined the effects of TCDD on the ability of DC to process and present antigen to antigen-specific T cells and to internalize latex beads. Additionally, the expression of costimulatory and adhesion molecules was examined on DC from TCDD-treated mice injected with allogeneic tumor cells. The ability of DC from C57Bl/6 mice to induce proliferation of keyhole limpet hemocyanin (KLH)-specific 10.5.17 T cells and production of IL-4 was not significantly altered by TCDD exposure, either when KLH was added in vitro or when the mice were injected with KLH prior to DC isolation. In contrast, ovalbumin (OVA) presentation by DC from TCDD-treated Balb/c mice induced enhanced proliferation of OVA-specific D011.10 T cells, although the production of IL-2 and IFN-gamma was not affected. Enhanced in vivo proliferation of adoptively transferred, CFSE-labeled DO11.10 T cells was also observed in TCDD-treated Balb/c mice that were challenged with OVA. TCDD treatment modulated the expression of major histocompatibility complex (MHC) class II, CD24, ICAM-1, CD40, and LFA-1 on splenic DC from C57Bl/6 mice injected with allogeneic tumor cells; however, the effects of TCDD were identical to changes seen previously in nonimmune mice, suggesting that these effects were not antigen-dependent. Finally, TCDD treatment did not affect the ability of splenic DC to internalize latex beads administered in vivo. Taken together, these results suggest that the activation-like changes induced in DC by TCDD do not suppress the ability of DC to process and present antigen, but may enhance their ability to provide activation signals to T cells. This, in turn, may alter the survival of the T cells, the DC, or both, and might lead to dysregulation of the immune response.

Exposure to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses the generation of T cell-dependent immunity, both humoral and cell-mediated. However, the mechanism of TCDD-induced immune suppression remains to be defined. We hypothesized that exposure to TCDD suppresses the activation of naive CD4(+) T cells and prevents their expansion and differentiation into effector T-helper cells capable of driving T cell-dependent immune responses. To test this hypothesis, we adoptively-transferred DO11.10 OVA-specific T-cell receptor (TCR) transgenic T cells into syngeneic recipients and used a TCR-specific monoclonal antibody to track the in vivo activation of naive CD4(+) T lymphocytes following exposure to OVA. The production of OVA-specific antibodies was suppressed in a dose-dependent manner in adoptively transferred mice that had been exposed to TCDD. Although TCDD exposure had little effect on the expansion or activation of the adoptively transferred, OVA-specific CD4(+) T cells, these cells disappeared from the spleen more rapidly in TCDD-treated mice and produced significantly decreased levels of the T cell-derived cytokines IL-2 and IL-10. There was also a trend towards reduced IFN-gamma and IL-4 production following in vitro re-stimulation. These data suggest that TCDD may interfere with the survival and/or differentiation of OVA-specific T-helper cells. These results demonstrate for the first time the potential of the DO11.10 adoptive transfer system to directly assess immunotoxic effects of xenobiotics on antigen-specific CD4(+) T cells in vivo.

The mechanism of arsine (AsH3) toxicity is not completely understood. In this investigation, we determined AsH3 and arsenite (AsIII) toxicity in Sprague Dawley rat blood, liver, and kidney. In all systems, there were dose- and time-dependent responses. Red blood cells were very susceptible to AsH3 toxicity. This was demonstrated by an immediate intracellular potassium loss and by hemolysis and lactate dehydrogenase (LDH) leakage that occurred by one h. AsIII concentrations up to 1 mM were not toxic to red blood cells using these indicators. Both AsH3 and AsIII produced toxicity in primary hepatocytes. Both produced significant LDH leakage and decreases in intracellular K+ by 5 h, but AsIII was more toxic than AsH3. At 24 h, both arsenic species showed similar toxicity. In renal cortical epithelial cells, AsH3 produced no effects on LDH and K+ over a 5-h period but produced significant LDH leakage by 24 h. In these cells, AsIII produced significant toxicity as early as in 3 h. These results showed that unchanged AsH3 produced toxicity in tissues, in addition to blood, and that toxicity of arsenicals is arsenic species- and tissue-dependent.

Recent developments in basic immunology have revealed the importance of glutathione (GSH) and cellular redox balance in the generation of an immune response. In the liver, it has been shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters cellular GSH and reactive oxygen intermediate (ROI) production. We have tested the hypothesis that TCDD mediates the suppression of the cytotoxic T lymphocyte (CTL) response to alloantigen by increasing oxidative stress. Total cellular GSH, GSSG, and GSH-protein adducts were analyzed by HPLC. Changes in intracellular GSH and ROI were simultaneously measured in isolated hepatocytes and individual subpopulations of spleen cells (CD4+, CD8+, B220+, and Mac-1+) following in vivo exposure to TCDD and antigenic challenge with P815 mastocytoma cells. Monochlorobimane was utilized to measure GSH levels, and two fluorescent probes were used to evaluate ROI levels: dichlorofluoroscein diacetate to monitor peroxides and dihydroethidine to assess superoxide anion. In hepatocytes, in vivo treatment with TCDD resulted in a transient, 2-fold increase in GSH, a 50% decrease in peroxide levels and a small (20-40%) decrease in superoxide anion levels. Although alloantigen challenge resulted in increased GSH and peroxide in spleen cells, in vivo exposure to TCDD had no effect on splenic ROI levels, nor did it consistently alter GSH levels in any subpopulation of spleen cells examined. Moreover, in vivo treatment with the antioxidant N-acetyl cysteine failed to affect the immune suppression caused by TCDD. These results suggest to us that although TCDD perturbs cellular redox balance in the liver, it does not exacerbate or diminish the normal increased GSH and ROI which occur in the spleen in response to antigenic challenge.

The impact of xenobiotics on intercellular adhesion, a fundamental biological process regulating most, if not all, cellular pathways, has been sparsely investigated. Cell-cell adhesion is regulated in the epithelium primarily by the E-cadherin/catenin complex. To characterize the impact of oxidative stress on the E-cadherin/catenin complex, precision-cut mouse liver slices were challenged with two model compounds for the generation of oxidative stress, diamide (DA; 25-250 microM) or t-butylhydroperoxide (tBHP; 5-50 microM), for 6 h. At the concentrations used, neither compound elicited cytotoxicity, as assessed by intracellular K+ content and leakage of lactate dehydrogenase into the culture media. However, a 25% reduction in non-protein sulfhydryl levels, an indication of oxidative perturbation, was seen in liver slices treated with DA or tBHP. Total protein expression of E-cadherin, beta-, or alpha-catenin was not affected by challenge with DA or tBHP. A decrease of beta-catenin in the SDS-soluble fraction of slices, an indicator of the formation of the adhesion complex, was observed. Additionally, a decrease in beta-catenin interactions with E-cadherin and alpha-catenin, as assessed by immunoprecipitation and Western blot analysis, was seen. Disruption of the E-cadherin/catenin complex by tBHP, but not DA, correlated with enhanced tyrosine phosphorylation of beta-catenin. These results suggest that noncytotoxic oxidative stress disrupts the E-cadherin/catenin cell adhesion complex in precision-cut mouse liver slices.