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A public appeal has been advanced by a large group of scientists, concerned that science has been misused in attempting to quantify and regulate unmeasurable hazards and risks.1 The appeal recalls that science is unable to evaluate hazards that cannot be measured, and that science in such cases should not be invoked to justify risk assessments in health, safety and environmental regulations. The appeal also notes that most national and international statutes delineating the discretion of regulators are ambiguous about what rules of evidence ought to apply. Those statutes should be revised to ensure that the evidence for regulatory action is grounded on the standards of the scientific method, whenever feasible. When independent scientific evidence is not possible, policies and regulations should be informed by publicly debated trade-offs between socially desirable uses and social perceptions of affordable precaution. This article explores the premises, implications and actions supporting the appeal and its objectives.

2, 2-Bis (bromomethyl)-1, 3-propanediol (BMP) is an extensively used brominated flame retardant found in urethane foams and polyester resins. In a two year dietary study conducted by the National Toxicology Program, BMP caused neoplastic lesions at multiple sites including the urinary bladder in both rats and mice. The mechanism of its carcinogenic effect is unknown. In the present study, using SV-40 immortalized human urothelial cells (UROtsa), endpoints associated with BMP induced DNA damage and oxidative stress were investigated. The effects of time (1–24 h) and concentration (5–100 μM) on BMP induced DNA strand breaks were assessed via the alkaline comet assay. The results revealed evidence of DNA strand breaks at 1 and 3h following incubation of cells with non-cytotoxic concentrations of BMP. Strand breaks were not present after 6h of incubation. Evidences for BMP associated oxidative stress include: an elevation of intracellular ROS formation as well as induction of Nrf2 and HSP70 protein levels. In addition, DNA strand breaks were attenuated when cells were pre-treated with N-acetyl-L-cysteine (NAC) and oxidative base modifications were revealed when a lesion specific endonuclease, human 8-hydroxyguanine DNA glycosylase 1 (hOGG1) was introduced into the comet assay. In conclusion, these results demonstrate that BMP induces DNA strand breaks and oxidative base damage in UROtsa cells. Oxidative stress is a significant, determinant factor in mediating these DNA lesions. These early genotoxic events may, in part, contribute to BMP-induced carcinogenesis observed in rodents.

In vitro exposure of Postnatal Day 4 (PND4) rat ovaries to the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys specifically primordial and primary follicles via acceleration of atresia. Because oocyte-expressed c-kit (KIT) plays a critical role in follicle survival and activation, a direct interaction of VCD with KIT as its mechanism of ovotoxicity was investigated. PND4 rat ovaries were cultured with and without VCD (30 μM) for 2 days. When assessed by Western analysis or mobility shift detection, phosphorylated KIT (pKIT) was decreased (P < 0.05) by VCD exposure, while total KIT protein was unaffected. Anti-mouse KIT2 (ACK2) antibody binds KIT and blocks its signaling pathways, whereas anti-mouse KIT 4 (ACK4) antibody binds KIT but does not block its activity. PND4 rat ovaries were incubated for 2 days with and without VCD with and without ACK2 (80 μg/ml) or ACK4 (80 μg/ml). ACK2 decreased pKIT; however, ACK4 had no effect. Conversely, ACK2 did not affect a VCD-induced decrease in pKIT, whereas ACK4 further reduced it. Because ACK2 and ACK4 (known to directly bind KIT) affect VCD responses, these results support the fact that VCD interacts directly with KIT. The effect of these antibodies on VCD-induced follicle loss was measured after 8 days of incubation. ACK2 further reduced (P < 0.05) VCD-induced follicle loss, whereas ACK4 did not affect it. These findings demonstrate that VCD induces ovotoxicity by direct inhibition of KIT autophosphorylation of the oocyte. The data also further support the vital function of KIT and its signaling pathway in primordial follicle survival and activation, as well as its role in VCD-induced ovotoxicity.

Ionic liquids (ILs) are a class of salts that are expected to be used as a new source of solvents and for many other applications. Our previous studies revealed that selected ILs, structurally related organic cations, are eliminated exclusively in urine as the parent compound, partially mediated by renal transporters. This study investigated the inhibitory effects of N-butylpyridinium chloride (NBuPy-Cl) and structurally related ILs on organic cation transporters (OCTs) and multidrug and toxic extrusion transporters (MATEs) in vitro and in vivo. After Chinese hamster ovary cells expressing rat (r) OCT1, rOCT2, human (h) OCT2, hMATE1, or hMATE2-K were constructed, the ability of NBuPy-Cl, 1-methyl-3-butylimidazolium chloride (Bmim-Cl), N-butyl-N-methylpyrrolidinium chloride (BmPy-Cl), and alkyl substituted pyridinium ILs to inhibit these transporters was determined in vitro. NBuPy-Cl (0, 0.5, or 2 mg/kg per hour) was also infused into rats to assess its effect on the pharmacokinetics of metformin, a substrate of OCTs and MATEs. NBuPy-Cl, Bmim-Cl, and BmPy-Cl displayed strong inhibitory effects on these transporters (IC(50) = 0.2-8.5 μM). In addition, the inhibitory effects of alkyl-substituted pyridinium ILs on OCTs increased dramatically as the length of the alkyl chain increased. The IC(50) values were 0.1, 3.8, 14, and 671 μM (hexyl-, butyl-, and ethyl-pyridinium and pyridinium chloride) for rOCT2-mediated metformin transport. Similar structurally related inhibitory kinetics were also observed for rOCT1 and hOCT2. The in vivo coadministration study revealed that NBuPy-Cl reduced the renal clearance of metformin in rats. These results demonstrate that ILs compete with other substrates of OCTs and MATEs and could alter the in vivo pharmacokinetics of such substrates.

Anti-Mullerian hormone (AMH) is produced by granulosa cells in primary to small antral follicles of the adult ovary and helps maintain primordial follicles in a dormant state. The industrial chemical, 4-vinylcyclohexene diepoxide (VCD) causes specific ovotoxicity in primordial and small primary follicles of mice and rats. Previous studies suggest that this ovotoxicity involves acceleration of primordial to primary follicle recruitment via interactions with the Kit/Kit ligand signaling pathway. Because of its accepted role in inhibiting primordial follicle recruitment, the present study was designed to investigate a possible interaction between AMH and VCD-induced ovotoxicity. Protein distribution of AMH was compared in neonatal and adult F344 rat ovaries. AMH protein was visualized by immunofluorescence microscopy in large primary and secondary follicles of the adult ovary, but in small primary follicles in neonatal rat ovaries. In cultured postnatal day (PND) 4 F344 rat ovaries, VCD exposure (30 μM, 2-8 days) decreased (P

4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that specifically destroys primordial and small primary follicles in the ovaries of rats and mice, is thought to target an oocyte-expressed tyrosine kinase receptor, Kit. This study compared the temporal effect of VCD on protein distribution of KIT and its downstream PIK3-activated proteins, AKT and FOXO3. Postnatal Day 4 Fischer 344 rat ovaries were cultured in control media ± VCD (30 μM) for 2-8 days (d2-d8). KIT, AKT, phosphorylated AKT, FOXO3, and pFOXO3 protein levels were assessed by Western blotting and/or immunofluorescence staining with confocal microscopy. Phosphorylated AKT was decreased (P < 0.05) in oocyte nuclei in primordial (39% decrease) and small primary (37% decrease) follicles within 2 days of VCD exposure. After d4, VCD reduced (P < 0.05) oocyte staining for KIT (primordial, 44% decrease; small primary, 39% decrease) and FOXO3 (primordial, 40% decrease; small primary, 36% decrease) protein. Total AKT and pFOXO3 were not affected by VCD at any time. Akt1 mRNA, as measured by quantitative RT-PCR, was reduced (P < 0.05) by 23% on d4 of VCD exposure, but returned to control levels on d6 and d8. VCD exposure reduced Foxo3a mRNA by 26% on d6 (P < 0.05) and by 23% on d8 (P < 0.1). These results demonstrate that the earliest observed effect of VCD is an inhibition of phosphorylation and nuclear localization of AKT in the oocyte of primordial and small primary follicles. This event is followed by reductions in KIT and FOXO3 protein subcellular distribution prior to changes in mRNA. Thus, these findings further support that VCD induces ovotoxicity by directly targeting the oocyte through posttranslational inhibition of KIT-mediated signaling components.

The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral follicles in rats and mice via apoptosis. Detoxification of VCD can occur through glutathione conjugation, catalyzed by glutathione S-transferase (GST) enzymes. Further, GST class pi (GSTp) can negatively regulate JNK activity through protein:protein interactions in extra-ovarian tissues. Dissociation of this protein complex in the face of chemical exposure releases the inhibition of pro-apoptotic JNK. Increased JNK activity during VCD-induced ovotoxicity has been shown in isolated ovarian small pre-antral follicles following in vivo dosing of rats (80 mg/kg/day; 15 days, i.p.). The present study investigated the pattern of ovarian GSTp expression during VCD exposure. Additionally, the effect of VCD on an ovarian GSTp:JNK protein complex was investigated. PND4 F344 rat ovaries were incubated in control medium {+-} VCD (30 {mu}M) for 2-8 days. VCD increased ovarian GSTp mRNA (P < 0.05) relative to control on d4-d8; whereas GSTp protein was increased (P < 0.05) on d6-d8. A GSTp:JNK protein complex was detected by immunoprecipitation and Western blotting in ovarian tissues. Relative to control, the amount of GSTp-bound JNK was increased (P = 0.09), while unbound JNK was decreased (P < 0.05) on d6 of VCD exposure. The VCD-induced decrease inmore » unbound JNK was preceded by a decrease in phosphorylated c-Jun which occurred on d4. These findings are in support of a possible dual protective role for GSTp in the rat ovary, consisting of metabolism of VCD and inhibition of JNK-initiated apoptosis.« less

Background: Contact hypersensitivity quantitative risk assessment (QRA) for fragrance ingredients is being used to establish new international standards for all fragrance ingredients that are potential skin sensitizers. Objective: The objective was to evaluate the retrospective clinical data on three fragrance ingredients in order to provide a practical assessment of the predictive value of the QRA approach. It is important to have data to assess that the methodology provides a robust approach for primary prevention of contact sensitization induction for fragrance ingredients identified as potential sensitizers. Methods: This article reviews clinical data for three fragrance ingredients cinnamic aldehyde, citral, and isoeugenol to assess the utility of the QRA approach for fragrance ingredients. Results: This assessment suggests that had the QRA approach been available at the time standards were established for these fragrance ingredients, the clinical response might have been noticeably improved. Prospectively, with the establishment of QRA-derived standards, there should be a continued downward trend in patch test-positive rates for cinnamic aldehyde, citral, and isoeugenol over time. Conclusion: While it is recognized that the availability of retrospective data is limited, a longitudinal review of these data gives confidence that the QRA approach should be an effective tool for primary prevention. This study also highlights the importance of continued active monitoring of clinical patch-test data for fragrance ingredients. (Less)

4-vinylcyclohexene diepoxide (VCD) is an ovotoxicant that specifically destroys primordial and small primary follicles in the ovaries of mice and rats. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA) is ovotoxic to all ovarian follicle classes. This study investigated phosphatidylinositol-3 kinase signaling involvement in VCD- and DMBA-induced ovotoxicity. Postnatal day (PND) 4 Fischer 344 (F344) rat whole ovaries were cultured for 2-12 days in vehicle control, VCD (30 microM), or DMBA (1 microM), +/-PI3 kinase inhibitor LY294002 (20 microM) or its inactive analog LY303511 (20 microM). Following culture, ovaries were histologically evaluated, and healthy follicles were classified and counted. PI3 kinase inhibition had no effect on primordial follicle number, but reduced (P0.05) small primary and larger follicles beginning on day 4. VCD caused primordial and small primary follicle loss (P0.05) beginning on day 6. With PI3 kinase inhibition, VCD did not affect primordial follicles (P0.05) at any time, but did cause loss (P0.05) of small primary follicles. DMBA exposure caused primordial and small primary follicle loss (P0.05) on day 6. Further, DMBA-induced primordial and small primary follicle loss was greater with PI3 kinase inhibition (P0.05) than with DMBA alone. These results support that (1) PI3 kinase mediates primordial to small primary follicle recruitment, (2) VCD, but not DMBA, enhances ovotoxicity by increasing primordial to small primary follicle recruitment, and (3) in addition to xenobiotic-induced ovotoxicity, VCD is also a useful model chemical with which to elucidate signaling mechanisms involved in primordial follicle recruitment.

Recent public concern has focused on potential reproductive and developmental effects from exposure to low levels of bisphenol A (BPA, CAS number 80-05-7). Two previous published reviews (; ) condu...

4-vinylcyclohexene diepoxide (VCD) specifically destroys small pre-antral follicles in the rodent ovary. VCD can be detoxified to an inactive tetrol by microsomal epoxide hydrolase (mEH), or by conjugation to glutathione (GSH) by glutathione S-transferase (GST). Formation of VCD-GSH adducts in the mouse ovary 4 h after VCD exposure (0.57 mmol/kg/day) has been demonstrated. Because the mouse ovary expresses both mEH and GST, expression of mEH and GST pi and mu during a time-course of VCD-induced ovotoxicity was evaluated in a neonatal mouse ovarian culture system. Ovaries from postnatal day 4 (PND4) B6C3F(1) mice were incubated with VCD (15 microM) for 2, 4, 6, 8, 10, 12, or 15 days. Following incubation, ovaries were histologically evaluated, or assessed for mRNA or protein expression. VCD did not cause follicle loss (p0.05) on days 2, 4, or 6 of culture. At days 8, 10, 12, and 15, VCD reduced (p0.05) both primordial and primary follicle numbers. Increased (p0.05) expression of mEH, GST pi and GST mu mRNA was detected after 4 days of VCD exposure. This expression was reduced on days 6 and 8, when follicle loss was underway, but increased (p0.05) after 10 days of exposure. mEH and GST pi proteins were elevated (p0.05) following 8 days of VCD-exposure however there was no increase in GST mu protein. These findings suggest that with continuous exposure to VCD, increased expression of detoxification enzymes may participate in retarding the onset of follicle loss, but that this loss cannot ultimately be prevented.

BACKGROUND The occupational chemical 4-vinylcyclohexene (VCH) has been shown to cause destruction of small pre-antral follicles in ovaries of mice. Further, its monoepoxide metabolites, 1,2-VCH epoxide, 7,8-VCH epoxide, and the diepoxide, VCD, have been shown to cause pre-antral follicle loss in rats as well as mice. Chemicals that destroy small pre-antral follicles are of concern to women because exposure can result in premature ovarian failure (early menopause). METHODS Studies working with these chemicals over the past decade have determined a number of aspects of the mechanism(s) of small pre-antral destruction, and a variety of questions have been answered. RESULTS Specifically, it has been determined that the diepoxide (VCD) is the bioactive form and it directly targets the ovary in mice and rats. Mice are more susceptible to VCH than rats because they are capable of its metabolic bioactivation. Follicle destruction by VCD is selective for primordial and primary follicles. Mechanistic studies in rats have determined that VCD causes ovotoxicity by accelerating the natural process of atresia (apoptosis) and this requires repeated exposures. Pro-apoptotic signaling events in the Bcl-2 and mitogen activated protein kinase families have been shown to be selectively activated in fractions of small pre-antral follicles (targets for VCD). Finally, a whole ovarian culture system using neonatal mouse and rat ovaries has been developed to expand the potential for more in depth investigations into ovotoxicity caused by VCD. CONCLUSIONS This article provides an overview of the questions asked and the approaches taken in studying VCH and VCD to support these conclusions. Birth Defects Res (Part B), 2007. © 2007 Wiley-Liss, Inc.

Repeated dosing with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively depletes small pre-antral follicles in the ovaries of rats and mice via apoptosis. The aryl hydrocarbon receptor (AhR) plays a role in mediating the effects of several xenobiotics. Therefore, this study was designed to investigate a potential role of the AhR in VCD-induced ovotoxicity. Female F344 rats, C57BL/6 mice, or AhR-deficient (-/-, AhRKO) mice were dosed daily (15 days) with vehicle, VCD (80 mg/kg, i.p.) and/or the AhR antagonist, alpha-naphthoflavone (ANF; 80 mg/kg, i.p.). Compared with controls, VCD caused a 60% reduction (P0.05) in primordial and primary follicles in mice and rats. Concurrent dosing with ANF protected against the VCD-induced follicle loss in rats, but not in mice. As with AhR-intact mice and rats, VCD induced a 70% loss (P0.05) of small pre-antral follicles in AhRKO mice. AhR mRNA expression was increased (P0.05) by VCD dosing in small pre-antral follicles isolated from ovaries of rats but not mice. AhR protein in rats was increased by VCD dosing in oocyte nuclei in primordial and primary follicles when measured by immunofluorescence and confocal microscopy. In rat small pre-antral follicles, apoptosis-associated caspase-3-like activity was increased (P0.05) by VCD treatment, decreased (P0.05) by ANF treatment, and unaffected by VCD plus ANF treatment. VCD had no effect on expression of GST Ya1 or GST Ya2 mRNA or CYP 1A1 protein in rats. Taken together, these findings demonstrate a difference between rats and mice in the potential involvement of AhR as related to VCD-induced ovotoxicity. Whereas, AhR appears to be involved in rats, no evidence for a similar role in mice was obtained. Overall, these findings point out that there can be mechanistic species differences in ovarian responses to xenobiotic chemicals.

A panel convened by the Harvard Center for Risk Analysis (HCRA) evaluated the weight of evidence for potential developmental and reproductive toxicity of bisphenol A (BPA, CASRN 80-05-7) in animals at doses well below the Lowest Observed Adverse Effect Level (LOAEL) of 50 mg/kg-day previously identified by the U.S. Environmental Protection Agency (US EPA) and even US EPA's reference dose (RfD) of 0.05 mg/kg-day. The effects are hypothesized to occur through an endocrine-modulating mode of action, specifically through estrogen receptors. The panel focused on potential male reproductive effects but also examined other endpoints possibly associated with hormone-like effects. The review considered studies published through April 2002. A formal deliberation framework focused on consistency, generalizability, and biological plausibility. The panel found no consistent affirmative evidence of low-dose BPA effects for any endpoint. Inconsistent responses across rodent species and strains made generalizabi...

4-Vinylcyclohexene (VCH), an occupational chemical, causes destruction of small preantral follicles (F1) in mice. Previous studies suggested that VCH is bioactivated via cytochromes P450 (CYP450) to the ovotoxic, diepoxide metabolite, VCD. Whereas hepatic CYP450 isoforms 2E1, 2A, and 2B can metabolize VCH, the role of ovarian metabolism is unknown. This study investigated expression of these isoforms in isolated ovarian fractions (F1, 25-100 microm; F2, 100-250 microm; F3, >250 microm; interstitial cells, Int) from B6C3F1 mice dosed daily (15 days; ip) with vehicle, VCH (7.4 mmol/kg/day) or VCD (0.57 mmol/kg/day). Ovaries were removed and either isolated into specific ovarian compartments for mRNA analysis, fixed for immunohistochemistry, or prepared for enzymatic assays. mRNA and protein for all isoforms were expressed/distributed in all ovarian fractions from vehicle-treated mice. In the targeted F1 follicles, VCH or VCD dosing increased (p < 0.05) mRNA encoding CYP2E1 (645 +/- 14% VCH; 582 +/- 16% VCD), CYP2A (689 +/- 8% VCH; 730 +/- 22% VCD), and CYP2B (246 +/- 7% VCH) above control. VCH dosing altered (p < 0.05) mRNA encoding CYP2E1 in nontargeted F3 follicles (168 +/- 7%) and CYP2A in Int (207 +/- 19%) above control. Immunohistochemical analysis revealed the greatest staining intensity for all CYP isoforms in the Int. VCH dosing altered (p < 0.05) staining intensity in Int for CYP2E1 (19 +/- 2.4% below control) and CYP2A (39 +/- 5% above control). Staining intensity for CYP2B was increased (p < 0.05) above control in granulosa cells of small preantral (187 +/- 42%) and antral (63 +/- 8%) follicles. Catalytic assays in ovarian homogenates revealed that CYP2E1 and CYP2B were functional. Only CYP2E1 activity was increased (149 +/- 12% above control; p < 0.05) by VCH dosing. The results demonstrate that mRNA and protein for CYP isoforms known to bioactivate VCH are expressed in the mouse ovary and are modulated by in vivo exposure to VCH and VCD. Interestingly, there is high expression of these isoforms in the Int. Thus, the ovary may contribute to ovotoxicity by promoting bioactivation of VCH to the toxic metabolite, VCD.

Repeated daily dosing of rats with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) causes selective depletion of the smallest preantral ovarian follicles (primordial and primary). These targeted populations are difficult to study because they comprise very little of the overall mass of ovarian tissue. Additionally, they are randomly distributed throughout the ovary. Therefore, a neonatal rat ovarian culture system containing predominantly primordial and primary follicles was developed and its susceptibility to VCD was assessed. The in vivo sensitivity of neonatal rats to VCD dosing was first confirmed by daily injection of VCD (80 mg/kg/day ip) on postnatal days (PND) 4-19. On PND 19, depletion of primordial and small primary follicles was evident. Ovarian cultures were then established utilizing a floating organ culture system to treat ovaries from PND 4 Fischer 344 rats in vitro. Initial follicle populations in cultured ovaries consisted of primordial (81%) and small primary (19%) follicles, whereas larger-sized preantral follicles had developed after 15 days in culture (67% primordial, 31% small primary, and 2% large primary). Cultured rat ovaries were sensitive to follicle depletion by incubation with VCD (> or =30 microM), and follicle loss occurred in a time-dependent manner (8-15 days). Evidence for apoptosis in VCD-exposed ovaries, as demonstrated in vivo, was obtained using immunohistochemistry. There was significantly more staining for apoptosis-associated active caspase-3 and TUNEL in ovaries incubated with VCD (30 microM, 15 days) compared with matched controls. These results demonstrate that small preantral follicles of cultured neonatal rat ovaries are sensitive to VCD exposure. The similarities between VCD's effects in vitro and in vivo demonstrate the usefulness of this system for future mechanistic studies related to ovarian follicle loss induced by VCD or other ovotoxic chemicals.

Previous studies have demonstrated that ovotoxicity induced in small preantral (primordial and primary) ovarian follicles by 4-vinylcyclohexene diepoxide (VCD) in rats is likely via acceleration of the normal process of atresia (apoptosis). This acceleration is associated with increased activities of caspase cascades, changes in subcellular distribution of Bcl-2 family members, and alteration of estrogen receptor-mediated signaling pathways. The present study was designed to investigate possible effects of VCD dosing on the mitogen-activated protein kinases (MAPK)/AP-1 signaling pathways in rat ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg i.p., 1 day—a time when ovotoxicity has not been initiated) or dosed daily for 10 or 15 days (80 mg/kg i.p.; 10 days—a time when the earliest signs of impending follicular destruction is seen, 15 days—a time when significant ovotoxicity is underway). Four hours following the final dose, ovaries and livers were collected. Ov...

Microsomal epoxide hydrolase (mEH) is involved in the detoxification of xenobiotics that are or can form epoxide metabolites, including the ovotoxicant, 4-vinylcyclohexene (VCH). This industrial chemical is bioactivated by hepatic CYP450 to the diepoxide metabolite, VCD, which destroys mouse small preantral follicles (F1). Since ovarian mEH may play a role in VCD detoxification, these studies investigated the expression and activity of mEH in isolated ovarian fractions. Mice were given 1 or 15 daily doses (ip) of VCH (7.4 mmol/kg/day) or VCD (0.57 mmol/kg/day); 4 h following the final dose, ovaries were removed, distinct populations of intact follicles (F1, 25-100 microm; F2, 100-250 microm; F3, > 250 microm) and interstitial cells (Int) were isolated, and total RNA and protein were extracted. Real-time polymerase chain reaction and the substrate cis-stilbene oxide (CSO; 12.5 microM) were used to evaluate expression and specific activity of mEH, respectively. Confocal microscopy evaluated ovarian distribution of mEH protein. Expression of mRNA encoding mEH was increased in F1 (410 +/- 5% VCH; 292 +/- 5% VCD) and F2 (1379 +/- 4% VCH; 381 +/- 11% VCD) follicles following repeated dosing with VCH or VCD. Catalytic activity of mEH increased in F1 follicles following repeated dosing with VCH/VCD (381 +/- 11% VCH; 384 +/- 27% VCD). Visualized by confocal microscopy, mEH protein was distributed throughout the ovary with the greatest staining intensity in the interstitial cells and staining in the theca cells that was increased by dosing (56 +/- 0.8% VCH; 29 +/- 0.9% VCD). We conclude that mEH is expressed and is functional in mouse ovarian follicles. Additionally,in vivo dosing with VCH and VCD affects these parameters.

Repeated dosing with 4-vinylcyclohexene diepoxide (VCD) accelerates atresia via apoptosis in primordial and primary follicles in ovaries of rats. The mechanisms that control atresia and VCD-induced toxicity are unknown; however, they could involve 17beta-E2. Atresia slows as animals enter puberty, whereas circulating E2 levels increase with the the onset of cyclicity. This inverse relationship suggests that E2 may be involved in the control of atresia. Therefore, this study was designed to determine whether treatment of immature rats with E2 could protect follicles normally destroyed by VCD-induced apoptosis. Female F344 rats were treated daily with E2, ER analogs, and/or VCD for 15 d. VCD alone caused a 50% reduction in primordial and primary follicles. Coinjection of E2 (0.1 mg/kg) and VCD (80 mg/kg) selectively protected primary follicles from VCD-induced follicle loss. This protection was mimicked by an ER agonist, genistein (0.1 mg/kg), and prevented by an ER antagonist, 4-hydroxytamoxifen (2 mg/kg). VCD treatment increased caspase-3-like activity, whereas concurrent treatment with genistein and VCD restored caspase-3-like activity to control levels. VCD treatment had no effect on circulating E2 levels, uterine weight, or E2 binding to the ER, nor could it directly displace E2 from ERbeta. These observations support the idea that ER-mediated protection against VCD-induced follicle toxicity is obtained by reducing apoptosis in small preantral follicles, although VCD does not appear to directly interact with ER.

A number of reports document that Fischer 344 (F344) rats are more susceptible to chemically induced liver injury than Sprague-Dawley (SD) rats. Cadmium (CdCl2), a hepatotoxicant that does not require bioactivation, was used to better define the biological events that are responsible for the differences in liver injury between F344 and SD rats. CdCl2 (3 mg/kg) produced hepatotoxicity in both rat strains, but the hepatic injury was 18-fold greater in F344 rats as assessed by plasma alanine aminotransferase (ALT) activity. This difference in toxicity was not observed when isolated hepatocytes were incubated with CdCl2 in vitro, indicating that other cell types contribute to Cd-induced hepatotoxicity in vivo. Indeed, the sieve plates of hepatic endothelial cells (EC) in F344 rats were damaged to a greater degree than EC in SD rats. Additionally, Kupffer cell (KC) inhibition reduced hepatotoxicity in both strains, suggesting that this cell type is involved in the progression of CdCl2-induced hepatotoxicity. Moreover, enhanced synthesis of heat shock protein 72 occurred earlier in the SD rat. Maximal levels of hepatic metallothionein (MT), a protein associated with cadmium tolerance, were greater in SD rats. These protective factors may limit CdCl2-induced hepatocellular injury in SD compared with F344 rats by reducing KC activation and the subsequent inflammatory response that allows for the progression of hepatic injury.

Previous studies have shown that 4-vinylcyclohexene diepoxide (VCD)-induced ovotoxicity in rats is likely caused by acceleration of the normal rate of atresia (apoptosis). VCD-induced ovotoxicity is specific for small preantral follicles and is associated with increased activity of caspase cascades. The present study was designed to investigate the alteration of expression and distribution of several Bcl-2 family member proteins induced by dosing of VCD in rat small ovarian follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., 1 day; a time when ovotoxicity is not initiated), or dosed daily for 15 days (80 mg/kg, i.p., 15 days; a time when significant ovotoxicity is underway). Four hours following the final dose, livers and ovaries were collected. Ovarian small (25–100 μm) and large (100–250 μm) preantral follicles were isolated, and subcellular fractions (cytosolic and mitochondrial) were prepared. Compared with controls, levels of the proapoptotic protein, Bad, were great...

Repeated daily dosing of rats with the occupational chemical 4- vinylcyclohexene or its diepoxide metabolite (VCD) for 15 days destroys the smallest ovarian follicles. VCD acutely reduced hepatic levels of the antioxidant, glutathione (GSH); therefore, these studies were designed to evaluate whether GSH concentrations mediate VCD-induced ovotoxicity. Immature female Fischer 344 rats were dosed once or daily for 15 days with VCD (0.57 mmol/kg, ip) or the GSH synthesis inhibitor buthionine sulfoximine (BSO, 2 mmol/kg, ip). Animals were euthanized 2, 6, or 26 h following a single dose, and 2 or 26 h following 15 days of daily dosing. Reduced (p < 0.05) hepatic GSH was seen within 2 h of a single dose of either VCD (51 +/- 5% of control) or BSO (42 +/- 9%), but only BSO reduced ovarian GSH (71 +/- 5% at 6 h, p = 0.05) as measured by HPLC. Within 26 h, GSH levels had returned to control levels with either treatment. Hepatic GSH levels were reduced (< 0.05) 2 h after 15 daily doses with BSO (42 +/- 5%) or VCD (70 +/- 4%), but only BSO decreased ovarian GSH (64 +/- 3%). GSH levels in 15-day tissues were similar to controls 26 h after the final dose. Neither BSO nor VCD increased hepatic or ovarian concentrations of the oxidized dimer of GSH (GSSG) or thiobarbituric acid-reactive substances (TBARS), indicators of oxidative stress. These results suggest these treatments did not cause an oxidative stress. Histological counts of ovarian small follicle numbers were reduced (p < 0.05) in 15-day VCD-treated rats, whereas BSO did not affect follicle numbers, even though BSO reduced ovarian GSH content. These results support the conclusion that alterations in ovarian GSH levels are not involved in VCD-induced ovotoxicity.

Previous studies have shown that ovotoxicity induced in rats by dosing with 4-vinylcyclohexene diepoxide (VCD) is likely via acceleration of the normal rate of atresia (apoptosis). The present study was designed to investigate the apoptosis-related caspase cascades as a component of this phenomenon in isolated ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg, i.p., on Day 1; a time when ovotoxicity has not been initiated), or dosed daily for 15 days (80 mg/kg, i.p., on Day 15; a time when significant ovotoxicity is underway). Ovaries were collected after the final dose. Small preantral follicles (25-100 microm in diameter) were isolated, cellular fractions were prepared, and cleavage activity or protein expression levels of caspases-3, -8, and -9 were measured. Cytosolic caspase-3 activity was increased in small follicles (P < 0.01) by VCD treatment (Day 1, 2.86 +/- 0.23; Day 15, 3.25 +/- 0.64, VCD/control, n = 3). This activation was not seen in large or antral follicles (not targeted by VCD). Procaspase-3 protein was increased(P < 0.05) by VCD treatment 212% over controls in small ovarian follicles in Day 15, but not Day 1-dosed rats. Immunofluorescence staining intensity was evaluated by confocal microscopy. Caspase-3 protein, located in the cytosolic compartment of oocytes and granulosa cells of preantral follicles in various stages of development, was selectively increased (P < 0.05) in primordial and small primary follicles from Day 15 VCD-dosed rats. Caspase-8 activity was increased in small follicles in Day 15, but not in Day 1-treated rats; whereas caspase-9 activity was increased by VCD on Day 1 in the mitochondrial fraction. Thus, these data provide evidence that accelerated atresia induced in small ovarian follicles in rats by VCD is associated with activation of a caspase-mediated cascade.

1,2-Dichlorobenzene (1,2-DCB) is a potent hepatotoxicant in male Fischer 344 (F344) rats and previous studies have suggested that reactive oxygen species may play a role in the development of hepatotoxicity. Since reactive oxygen species can damage lipid membranes, this study was conducted to determine the extent of lipid peroxidation after administration of 1,2-DCB by immunohistochemical analysis of 4-hydroxynonenal (4-HNE) protein adduct formation in liver and conjugated diene formation in liver and serum. The contribution of Kupffer cells to the lipid peroxidation was also investigated. Male F344 rats were administered 1,2-DCB (3.6 mmol/kg ip in corn oil) and killed at selected times between 3 and 48 h. Time course studies revealed the greatest abundance of 4-HNE protein adducts in the centrilobular regions of the liver 24 h after 1,2-DCB administration, with much lower levels at 16 h. Adducts were present in necrotic and vacuolized centrilobular hepatocytes of 1,2-DCB treated rats but not in livers of controls. Further, conjugated dienes were significantly increased in liver and serum 16 and 24 h after 1,2-DCB administration, peaking at 24 h. These data correlated with hepatocellular injury, determined by serum alanine aminotransferase activity and histopathological evaluation, which was markedly elevated within 16 h and peaked at 24 h. When rats were pretreated with gadolinium chloride (GdCl 3 ; 10 mg/kg iv 24 h prior to 1,2-DCB), an inhibitor of Kupffer cells, hepatotoxicity was decreased by 89 and 86%, at 16 and 24 h, respectively. Conjugated diene concentrations were decreased to control values at these times after 1,2-DCB administration. Moreover, no 4-HNE protein adducts were detected in livers of 1,2-DCB-treated rats pretreated with GdCl 3 . Finally, Kupffer cells isolated from 1,2-DCB-treated rats produced significantly more superoxide anion than Kupffer cells isolated from vehicle controls. These data, along with previous findings, suggest that lipid peroxidation associated with 1,2-DCB is mediated in part by Kupffer cell-derived reactive oxygen species.

Exposure of humans to toxic metals and metalloids is a major environmental problem. Many metals, such as cadmium, can be hepatotoxic. However, the mechanisms by which metals cause acute hepatic injury are in many cases unknown. Previous reports suggest a major role for inflammation in acute cadmium induced hepatotoxicity. In initial experiments we found that a non-hepatotoxic dose of cadmium chloride (CdCl 2 ; 2.0 mg/kg, i.v.) markedly increased the clearance rate of colloidal carbon from the blood, which is indicative of enhanced phagocytic activity by Kupffer cells (resident hepatic macrophages). Thus, the objective these studies was to determine the involvement of Kupffer cells in cadmium induced liver injury by inhibiting their function with gadolinium chloride (GdCl 3 ). Male Sprague-Dawley rats were administered GdCl 3 (10 mg/kg, i.v.) followed 24 h later by a single dose of CdCl 2 (3.0 and 4.0 mg/kg, i.v.). Twenty four hours after CdCl 2 administration animals were killed and the degree of liver toxicity was assessed using plasma alanine aminotransferase (ALT), as well as light microscopy. Cadmium chloride administration produced multifocal hepatocellular necrosis and increased plasma ALT activity. Pretreatment with GdCl 3 significantly reduced both the morphological changes and hepatic ALT release caused by CdCl 2 . However, the protection was specific to the liver, and did not alter CdCl 2 induced testicular injury, as determined by histopathological damage. In many cases, the inducible cadmium-binding protein, metallothionein (MT) is often an essential aspect of the acquisition of cadmium tolerance in the liver. Although cadmium caused a dramatic induction of hepatic MT (32-fold), GdCl 3 caused only a minor increase (2-fold). Combined CdCl 2 and GdCl 3 treatment did not induce levels to an extent greater than CdCl 2 alone. As expected, GdCl 3 also caused a slight increase in the amount of cadmium associated with the liver. In cultured hepatocytes isolated from GdCl 3 pretreated rats, CdCl 2 induced cytotoxicity was not significantly altered compared to control hepatocytes, indicating that the mechanism of tolerance required the presence of other cell types. Thus, GdCl 3 attenuation of CdCl 2 induced hepatotoxicity does not appear to be caused by increased tissue MT content or a decreased susceptibility of hepatocytes to cadmium. From these data, we concluded that tolerance to cadmium induced hepatotoxicity involves the inhibition of Kupffer cell function which results in a decreased inflammatory response and an altered progression of hepatic injury. These data further indicate that Kupffer cell function is critical to cadmium induced hepatocellular necrosis.

The reaction of peroxynitrite with gamma-tocopherol (gamma-TH) in a methanol/potassium phosphate buffer solution results in the formation of four major products, which were identified as 2,7,8-trimethyl-2-(4,8,12-trimethyldecyl)-5-nitro-6-chromanol++ + (NGT), 2,7,8-trimethyl-2-(4,8,12-trimethyldecyl)-5,6-chromaquinone (tocored), and two diastereomers of 8a-(hydroxy)-gamma-tocopherone. NGT was the major product formed in these reactions, and its formation was modestly increased by increasing amounts of Fe(3+)-EDTA. Tocored and NGT also were formed when gamma-TH was exposed to 3-morpholinosydnonimine (SIN-1), a compound that decomposes to form peroxynitrite. When gamma-TH reacted with the nitrating agent NO2+BF4- in acetonitrile or methanol/potassium phosphate buffer, NGT and tocored also were formed, but the major product detected was gamma-tocopherol quinone (gamma-TQ). This product was not detected in reactions involving peroxynitrite. Oxidation of gamma-TH by peroxynitrite involves nitration and electron transfer reactions. Since the product distribution in oxidations with NO2+BF4- differed substantially from that in oxidations with peroxynitrite and SIN-1, NO2+ appeared not to be the principal species involved in NGT formation. Nitration of gamma-TH may involve either peroxynitrite or some peroxynitrite-derived oxidant other than NO2+. Because of its stability and formation as a novel product of the reaction between gamma-TH with peroxynitrite, NGT may be a useful in vivo marker for peroxynitrite interactions with lipid structures that contain gamma-TH.

7-Acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN ) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyran (HHCB) are polycyclic musks widely used as fragrance ingredients in consumer products. Because their metabolic fate following systemic exposure is not fully characterized, disposition and excretion of 14C-AHTN- and 14C-HHCB-derived radioactivity were studied in Sprague-Dawley rats and domestic pigs following a single intravenous dose. Rats administered with AHTN or HHCB excreted 21% or 28% of the radioactivity in urine and 67% or 61% in feces, respectively, within 7 days. In pigs administered AHTN or HHCB, 86% or 74% of the dose was excreted in the urine, and 12% or 15% in feces, respectively, during the 14-day collection period. Radioactivity in the whole blood and plasma of both species and tissues of rats declined steadily until the end of the study (28 days) for both the materials. Radioactivity in rat adipose tissue reached peak at 2 hours after dosing, decreasing steadily thereafter. Radioactivity in pig blood declined rapidly from 70 ng equivalents/g at 10 minutes to 1 ng equivalent/g or less by 28 days after administration of either AHTN or HHCB. Radioactivity in pig skin and adipose tissue decreased to below the limit of detection by 28 days for both the materials. Thin-layer chromatography showed multiple radioactive components in both species’ urine after administration of either material. Components found in the urine of the 2 species were qualitatively similar but quantitatively different. Both AHTN and HHCB were completely metabolized and excreted. No unchanged parent compound was detected in rat or pig urine.

Pretreatment of large doses of vitamin A (VA) is known to potentiate the hepatotoxicity of carbon tetrachloride. Therefore the effects of 1-day VA pretreatment on VDC hepatotoxicity was examined bothin vivoand in anin vitrosystem of precision-cut rat liver slices. Male Sprague–Dawley rats were pretreated with 250,000 IU/kg VA by oral gavage. After 24 hr rats were administered 50, 100, or 200 mg/kg VDC ip. Precision-cut liver slices were prepared from VA pretreated rats 24 hr later and the liver slices were exposed for 2–8 hr to 0.025–1.0 μl VDC evaporated into the gas phase of the incubation vials. VA pretreatment resulted in an enhancement of VDC toxicity, bothin vivoandin vitro.There was a dose-dependent increase in plasma ALT 24 hr after VDC treatment of rats and an increase in K+leakage from liver slices after VDC exposure. Histological analysis of the liver or the liver slices revealed that VA + VDC treatment resulted in centrilobular necrosis of the liver. When GdCl3(10 mg/kg iv) was administered just before VA pretreatment of rats, VDC toxicity was partially reversed as observed by a decrease in ALTin vivoand a decrease in the loss of K+in vitro.These results indicated that Kupffer cells, the resident macrophages of the liver, were partially responsible for the VA-potentiated VDC hepatotoxicity. One-day pretreatment of VA induced cytochrome P450IIE1 protein content as well as its enzymatic activity as measured byp-nitrophenol hydroxylation. Because VDC is bioactivated by cytochrome P450IIE1, the increase in VDC hepatotoxicity after VA may be due to an increased bioactivation of VDC in the liver and in precision-cut liver slices. Thus, more than one mechanism may be involved in the VA enhancement of VDC hepatotoxicity. .

Vinylcyclohexene diepoxide (VCD) produces ovarian toxicity in female mice and rats, whereas testicular damage occurs only in mice. The objectives of these studies were to determine the target cell(s) and spermatogonial survival following VCD administration. In addition, the effects of 4-vinylcyclohexene (VCH) and two epoxide metabolites, vinylcyclohexene 1,2-monoepoxide and VCD were compared. Male mice were dosed daily with VCD (320 mg/kg/d, i.p.) and killed at 5, 10, 15, 20, 25, or 30 d. Two groups were dosed daily for 30 d and allowed to recover for 30 or 60 d. Decreases in testis weight began at 5 d and continued to 30 d. These decreases corresponded to progressive necrosis of germ cells. After 5 d of VCD, there was loss of Type I and B spermatogonia in Stages II to VI and of preleptotene spermatocytes in Stages VI to VIII. After 30 d of dosing, seminiferous tubules were devoid of germ cells except for spermatogonial stem cells. Following 30 d of recovery, 100% of the seminiferous tubules were repopulated. Epididymal spermatozoa were present after 60 d of recovery. Increasing doses of VCD (0 to 320 mg/kg/d) resulted in increasing testicular toxicity. Neither VCH (800 mg/kg, i.p.) nor VCM (200 mg/kg, i.p.) caused testicular damage. VCD administration initially results in destruction of spermatogonia and spermatocytes, which are undergoing DNA synthesis and cell replication, followed by loss of maturing cells. Neither VCH nor VCM caused testicular germ cell destruction, although all three compounds destroy germ cells in female mice. Therefore, further investigation will be necessary to understand these differences in chemical-induced toxicity between ovaries and testes.

The Interactions between Retinol and Five Different Hepatotoxicants in the Swiss Webster Mouse. Rosengren, R. J., Sauer, J.-M., Hooser, S. B., and Sipes, I. G. (1995). Fundam. Appl. Toxicol. 25, 281-292. The interactive effects between retinol and various hepatotoxicants (allyl alcohol, acetaminophen, carbon tetrachloride, D-galactosamine, and phalloidin) were studied in the male Swiss Webster mouse. The mice were administered retinol at 75 mg/kg/day (or the vehicle of retinol) by oral gavage for 7 days. Hepatoxicity produced by the chemicals was determined by plasma alanine aminotransferase (ALT) activity and histopathology. After 7 days of retinol pretreatment, the hepatotoxicities of allyl alcohol, acetaminophen, and galactosamine were potentiated. Interestingly, the hepatotoxicity of carbon tetrachloride and phalloidin was protected by identical retinol pretreatment. Microscopic examination of histologic liver sections demonstrated the specific hepatic necrosis associated with each individual chemical and confirmed the ALT values obtained. Once an interaction between retinol and the five hepatotoxicants was established, the duration of retinol pretreatment necessary to elicit an interaction was determined for each hepatotoxicant. Results demonstrated that the duration of retinol pretreatment was specific for each hepatotoxicant. The accumulation of retinoids in the liver during retinol pretreatment was determined using high-performance liquid chromatography analysis. Significant increases in the basal liver levels of retinol and retinyl palmitate were seen within 1 to 3 days of retinol treatment compared to control. Retinol pretreatment resulted in potentiation or protection of specific hepatotoxicant-induced liver damage. Currently, studies are being conducted which probe into the mechanisms of these interactions.

In vitro exposure of Postnatal Day 4 (PND4) rat ovaries to the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys specifically primordial and primary follicles via acceleration of atresia. Because oocyte-expressed c-kit (KIT) plays a critical role in follicle survival and activation, a direct interaction of VCD with KIT as its mechanism of ovotoxicity was investigated. PND4 rat ovaries were cultured with and without VCD (30 μM) for 2 days. When assessed by Western analysis or mobility shift detection, phosphorylated KIT (pKIT) was decreased (P < 0.05) by VCD exposure, while total KIT protein was unaffected. Anti-mouse KIT2 (ACK2) antibody binds KIT and blocks its signaling pathways, whereas anti-mouse KIT 4 (ACK4) antibody binds KIT but does not block its activity. PND4 rat ovaries were incubated for 2 days with and without VCD with and without ACK2 (80 μg/ml) or ACK4 (80 μg/ml). ACK2 decreased pKIT; however, ACK4 had no effect. Conversely, ACK2 did not affect a VCD-induced decrease in pKIT, whereas ACK4 further reduced it. Because ACK2 and ACK4 (known to directly bind KIT) affect VCD responses, these results support the fact that VCD interacts directly with KIT. The effect of these antibodies on VCD-induced follicle loss was measured after 8 days of incubation. ACK2 further reduced (P < 0.05) VCD-induced follicle loss, whereas ACK4 did not affect it. These findings demonstrate that VCD induces ovotoxicity by direct inhibition of KIT autophosphorylation of the oocyte. The data also further support the vital function of KIT and its signaling pathway in primordial follicle survival and activation, as well as its role in VCD-induced ovotoxicity.

Simultaneous administration of trichloroethylene (TCE), at an oral dose of 0.5 ml/kg, resulted in a marked potentiation of liver injury caused by an oral dose of carbon tetrachloride (CCl4, 0.05 ml/kg). Hepatic glutathione levels were depressed at 24 hr only in the rats given TCE and CCl4. Using serum enzyme (ALT and SDH) as indicators of hepatotoxicity, potentiation of CCl4-injury was most apparent at 24 hr. Upon histological examination of H&E stained liver sections, the differences between livers obtained from TCE and CCl4-treated rats versus CCl4-treatcd rats were most apparent at later time points (48 and 72 hr). At 48 hr after CCl4, livers showed a distinctive and uniform pattern of injury with regeneration features predominating over necrosis. At this time, livers from TCE and CCl4-treated rats were characterized by extensive zone 3 coagulative necrosis. Inflammatory infiltrations were less prominent. At 72 hr, morphological features of livers from TCE and CCl4 rats were similar to those from rats given CCl4 alone at 48 hr. From the results obtained, it appears that the regenerative activity of the liver may be delayed in rats simultaneously administered TCE and CCl4 as compared to rats administered only CCl4.

Resuscitation from hemorrhagic shock causes hepatic injury that is similar to the hypoxic injury caused by reperfusion after ischemia. This study was designed to describe the relationship between severe hemorrhagic shock, hepatic injury, and lipid peroxidation. Fasted Sprague-Dawley rats underwent shock (mean arterial pressure 40 +/- 5 mm Hg) for two hours followed by reinfusion of shed blood. Serum ALT levels increased during shock and gradually continued to increase for 24 hours after resuscitation. Lipid peroxidation was assessed by quantification of exhaled ethane and by liver content of thiobarbituric acid reactive substances (TBARS). Expired ethane was increased both during shock and after resuscitation. Hepatic content of TBARS remained at baseline levels during shock, but increased after resuscitation. The results suggest that severe, non-fatal hemorrhagic shock and resuscitation produces a modest hepatic injury that is accompanied by lipid peroxidation in the liver.

Contact hypersensitivity quantitative risk assessment (QRA) for fragrance ingredients is being used to establish new international standards for all fragrance ingredients that are potential skin sensitizers.The objective was to evaluate the retrospective clinical data on three fragrance ingredients in order to provide a practical assessment of the predictive value of the QRA approach. It is important to have data to assess that the methodology provides a robust approach for primary prevention of contact sensitization induction for fragrance ingredients identified as potential sensitizers.This article reviews clinical data for three fragrance ingredients-cinnamic aldehyde, citral, and isoeugenol-to assess the utility of the QRA approach for fragrance ingredients.This assessment suggests that had the QRA approach been available at the time standards were established for these fragrance ingredients, the clinical response might have been noticeably improved. Prospectively, with the establishment of QRA-derived standards, there should be a continued downward trend in patch test-positive rates for cinnamic aldehyde, citral, and isoeugenol over time.While it is recognized that the availability of retrospective data is limited, a longitudinal review of these data gives confidence that the QRA approach should be an effective tool for primary prevention. This study also highlights the importance of continued active monitoring of clinical patch-test data for fragrance ingredients.

2,2-Bis(bromomethyl)-1,3-propanediol (BMP) is a brominated flame retardant used in unsaturated polyester resins. In a 2-year bioassay BMP was shown to be a multisite carcinogen in rats and mice. Because glucuronidation is the key metabolic transformation of BMP by rats, in this study the in vitro hepatic glucuronidation of BMP was compared across several species. In addition, the glucuronidation activities of human intestinal microsomes and specific human hepatic UDP-glucuronosyltransferase (UGT) enzymes for BMP were determined. To explore other possible routes of metabolism for BMP, studies were conducted with rat and human hepatocytes. Incubation of hepatic microsomes with BMP in the presence of UDP-glucuronic acid resulted in the formation of a BMP monoglucuronide. The order of hepatic microsomal glucuronidation activity of BMP was rats, mice >> hamsters > monkeys >>> humans. The rate of glucuronidation by rat hepatic microsomes was 90-fold greater than that of human hepatic microsomes. Human intestinal microsomes converted BMP to BMP glucuronide at a rate even lower than that of human hepatic microsomes. Among the human UGT enzymes tested, only UGT2B7 had detectable glucuronidation activity for BMP. BMP monoglucuronide was the only metabolite formed when BMP was incubated with suspensions of freshly isolated hepatocytes from male F-344 rats or with cryopreserved human hepatocytes. Glucuronidation of BMP in human hepatocytes was extremely low. Overall, the results support in vivo studies in rats in which BMP glucuronide was the only metabolite found. The poor glucuronidation capacity of humans for BMP suggests that the pharmacokinetic profile of BMP in humans will be dramatically different from that of rodents.

In vitro studies using rat and human hepatic microsomes have shown that the halogenated hydrocarbon 1,2,3-trichloropropane (TCP) is bioactivated to the direct acting mutagen 1,3-dichloroacetone (DCA). The presence of DCA in microsomal incubations was confirmed by gas chromatography-mass spectrometry. DCA formation was totally dependent on the presence of NADPH. The rate of DCA formation using rat and human microsomes was 0.268 ± 0.029 and 0.026 ± 0.006 nmol/min/mg protein ± SE, respectively. When hepatic microsomes were isolated from rats pretreated with the cytochrome P-450 inducers, phenobarbital, and dexamethasone, 24- and 2.5-fold increases, respectively, in the rate of DCA production, were observed. Pretreatment with β-naphthoflavone resulted in a 50% inhibition in DCA formation. The inhibitors of cytochromes P-450, SKF 525-A and 1-aminobenzotriazol, produced 85 and 70% inhibitions of DCA formation, respectively. When alcohol dehydrogenase and NADH were added to microsomal incubations, two TCP-related alcohols, 1,3-dichloro-2-propanol and 2,3-dichloropropanol, were formed. These alcohols are products of the initial microsomal metabolites, DCA and 2,3-dichloropropanal. [ 14 C]TCP equivalents bound covalently to rat hepatic microsomal protein. This binding was increased 8-fold when hepatic microsomes from phenobarbital pretreated rats were used. The addition of either glutathione or N -acetylcysteine to the incubations completely inhibited this binding. In the presence of N -acetylcysteine, 1,3-(2-propanone)-bis- S -( N -acetylcysteine) (PDM) was the only N -acetylcysteine conjugate detected. It represented 87% of TCP microsomal metabolism. The formation of PDM implicates DCA as the major microsomal protein-binding metabolite of TCP. The formation of DCA, a direct-acting mutagen, may be responsible for the mutagenicity of TCP in systems using rat hepatic microsomes. Its role in the tumorigenicity and carcinogenicity of TCP remains to be established.

Previous studies have demonstrated that various compounds, including the common groundwater contaminants trichloroethylene (TCE) and chloroform (CHCl3), can produce a synergistic toxic response when coadministered with the model hepatotoxicant carbon tetrachloride (CCl4). This phenomenon has not, however, been demonstrated following administration of these compounds in drinking water. Initial experiments indicated that Fischer 344 (F-344) rats were significantly more sensitive to these effects than the more commonly utilized Sprague-Dawley strain. To establish the suitability of this strain as a model, a variety of indicators of hepatotoxicity was evaluated and compared to histological evidence of injury 24 hr after dosing with CCl4 or a combination of CCl4 + TCE. Plasma alanine aminotransferase (ALT) activity was the most reliable indicator of hepatic injury and was well-correlated with the histologic data. Dose-response studies utilizing simultaneous ip dosing confirm the sensitivity of the F-344 rat, demonstrating synergistic toxicity at doses as low as 0.165 mmol/kg of CCl4 and 0.6 mmol/kg of TCE. Synergism was also detected following simultaneous ip administration of 1 mmol/kg CCl4 and 0.5 mmol/kg of CHCl3. To evaluate the effects of drinking water exposure, rats were pretreated for 3 days with solutions containing TCE (0-40 mM) or CHCl3 (0-8 mM) stabilized with 1% Emulphor (EL-620P) as their only source of fluids. A single, ip dose of CCl4 (1 mmol/kg) was then administered and 24 hr later animals were killed for examination of liver histology and determination of ALT activity. Although none of the pretreatments were detectably hepatoxic, rats which drank 15 and 40 mM TCE or 8 mM CHCl3 exhibited an enhanced response to CCl4.

Assessment of ovarian toxicity by follicle quantitation and morphometric analysis of serial sections is time consuming and expensive. This report compares the estimation of follicle number obtained from counting oocytes in serial sections or 5 random sections of mouse ovaries. Ovaries were obtained from C57BL/6N and B6C3F1 mice treated with ovarian toxicants. C57BL/6N mice were treated with cyclophosphamide (0, 75, 200, and 500 mg/kg, ip) and killed at 24, 72, and 168 h. B6C3F1 mice were treated daily, ip, with 4-vinylcyclohexene (0, 100, 400, and 800 mg/kg/day for 30 days), vinylcyclohexene diepoxide (0, 10, 40, and 80 mg/kg/day for 30 days), or benzo(a)pyrene (100 mg/kg, single dose) and killed on day 31. Ovarian serial sections were prepared and oocytes counted in every tenth section. When serial sections were evaluated, 30 to 60 sections were counted. Random section counting involved randomly selecting 5 sections from the 30 to 60 sections previously counted by the serial method. Chemically-induced follicle loss was evident by the reduction in follicle counts relative to control animals when estimating follicle number using serial or random section counting. Furthermore, a linear regression analysis of follicle counts over all treatment groups showed that the highest correlation between random and serial section counting and was for primordial follicles. Correlation coefficients ( R 2 ) for each follicle type were: primordial-C57BL/6N 0.82, B6C3F1 0.86; growing-C57BL/6N 0.16, B6C3F1 0.46; antral-C57BL/6N 0.07, B6C3F1 0.11. Although other screens may need to be developed for growing and antral follicles, primordial follicle toxicity is adequately defined by counting 5 random sections.

2,2-Bis(bromomethyl)-1,3-propanediol (BMP) is a brominated flame retardant, previously shown to be a multisite carcinogen in experimental animals. Studies were performed to characterize the dispositional and metabolic fate of BMP after oral or intravenous administration to male Fischer-344 rats. After a single oral administration of [14C]BMP (10 or 100 mg/kg) >80% of the low dose and 48% of the high dose were excreted by 12 h in the urine predominantly as a glucuronide metabolite. After repeated daily oral doses for 5 or 10 days, route and rate of elimination were similar to those obtained after single administrations of BMP. In all studies, the radioactivity recovered in feces was low (50% within 6 h), but it underwent enterohepatic recycling with subsequent elimination in the urine. These data indicate that the extensive extraction and rapid glucuronidation by the liver limits exposure of internal tissues to BMP by greatly reducing its systemic bioavailability after oral exposure.

The polycyclic aromatic hydrocarbon 7, 12-dimethylbenz[a]anthracene, (DMBA), targets and destroys all follicle types in rat and mouse ovaries. DMBA requires bioactivation to DMBA-3,4-diol-1,2-epoxide for ovotoxicity via formation of the intermediate, DMBA-3,4-diol (catalyzed by microsomal epoxide hydrolase; mEH). mEH was shown to be involved in DMBA bioactivation for ovotoxicity induction in B6C3F(1) mouse ovaries. The current study compared DMBA and DMBA-3,4-diol mediated ovotoxicity, and investigated mEH involvement in DMBA-3,4-diol bioactivation in Fischer 344 (F344) rat ovary. F344 postnatal day (PND) 4 rat ovaries were cultured in vehicle control or media containing 1) DMBA or DMBA-3,4-diol (12.5 nM - 1 muM; 15 days); 2) DMBA (1 muM; 6 h - 15 days); and 3) DMBA (1 muM) or DMBA-3,4-diol (75 nM)+/-the mEH activity inhibitor cyclohexene oxide (CHO; 2 mM; 4 days). Ovaries were histologically evaluated and mEH mRNA and protein were measured by reverse transcriptase PCR or Western blotting, respectively. Ovotoxicity following 15 days of culture occurred (P

Recent public concern has focused on potential reproductive and developmental effects from exposure to low levels of bisphenol A (BPA, CAS number 80-05-7). Two previous published reviews (Gray et al., 2004a; Goodman et al., 2006) conducted weight-of-evidence evaluations of in vivo reproductive/developmental toxicity from BPA exposureor = 5 mg/kg-d based on studies published through February 2006. Here, an update of those analyses presents additional relevant studies that were published through July 25, 2008, and a weight-of-evidence analysis of the studies evaluated in all three reviews. As with the earlier literature, positive findings: (1) are countered by null findings in more numerous studies; (2) have not been replicated; (3) do not exhibit coherence and plausibility; (4) do not show consistency across species, doses, and time points; and/or (5) were from studies using non-oral exposure routes. Owing to the lack of first-pass metabolism, results from non-oral studies are of limited relevance to human exposure. Exposure levels in most of the low-dose oral and non-oral animal studies are generally much higher than those experienced by even the most exposed people in the general population. The weight of evidence does not support the hypothesis that low oral doses of BPA adversely affect human reproductive and developmental health.

Females are born with a finite number of primordial follicles. 4-Vinylcyclohexene diepoxide (VCD) is a metabolite formed by epoxidation of 4-vinylcyclohexene (VCH) via its two monoepoxides 1,2- and 7,8-4-vinylcyclohexene monoepoxide (VCM). VCD specifically destroys small preantral (primordial and small primary) follicles in the rodent ovary. The phase I enzyme, cytochrome P450 isoform 2E1 (CYP2E1) is involved in ovarian metabolism of VCM to VCD. Further, microsomal epoxide hydrolase (mEH) can detoxify VCD to an inactive tetrol (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane). This study evaluated the effects of VCD-induced ovotoxicity on mEH in CYP2E1+/+ and −/− mice (129S1/SvImJ background strain) using a postnatal day 4 mouse whole ovary culture system. The hypothesis of our study is that there is a relationship between CYP2E1 and mEH gene expression in the mouse ovary. Relative to control, VCD exposure caused follicle loss (p < 0.05) in ovaries from both genotypes; however, after 15 days, this loss was greater (p < 0.05) in CYP2E1+/+ ovaries. In a time course (2–15 days), relative to control, VCD (5μM) caused an increase (p < 0.05) in mEH mRNA by 0.5-fold (day 10) and 1.84-fold (day 15) in CYP2E1−/− but not +/+ ovaries. 7,12-Dimethylbenz[a]anthracene (DMBA) also destroys ovarian follicles but, unlike VCD, is bioactivated by mEH to an ovotoxic 3,4-diol-1,2-epoxide metabolite. Incubation of ovaries in increasing concentrations of DMBA (0.5–1μM, 15 days) resulted in greater (p < 0.05) follicle loss in CYP2E1−/−, relative to +/+ ovaries. With greater mEH (CYP2E1−/−), increased follicle loss with DMBA (bioactivation) and decreased follicle loss with VCD (detoxification) support that ovarian expression of CYP2E1 and mEH may be linked.