Development of a single-meal fish consumption advisory for methyl mercury.

Methyl mercury (meHg) contamination of fish is the leading cause of fish consumption advisories in the United States. These advisories have focused upon repeated or chronic exposure, whereas risks during pregnancy may also exist from a single-meal exposure if the fish tissue concentration is high enough. In this study, acute exposure to meHg from a single fish meal was analyzed by using the one-compartment meHg biokinetic model to predict maternal hair concentrations. These concentrations were evaluated against the mercury hair concentration corresponding to the U.S. Environmental Protection Agency's reference dose (RfD), which is intended to protect against neurodevelopmental effects. The one-compartment model was validated against blood concentrations from three datasets in which human subjects ingested meHg in fish, either as a single meal or multiple meals. Model simulations of the single-meal scenario at different fish meHg concentrations found that concentrations of 2.0 ppm or higher can be associated with maternal hair concentrations elevated above the RfD level for days to weeks during gestation. A single-meal fish concentration cutoff of > or = 2.0 ppm is an important consideration, especially because this single high exposure event might be in addition to a baseline meHg body burden from other types of fish consumption. This type of single-meal advisory requires that fish sampling programs provide data for individual rather than composited fish, and take into account seasonal differences that may exist in fish concentrations.

A physiologically-based pharmacokinetic model assessment of methyl t-butyl ether in groundwater for a bathing and showering determination.

Methyl t-butyl ether (MTBE) is a gasoline additive that has appeared in private wells as a result of leaking underground storage tanks. Neurological symptoms (headache, dizziness) have been reported from household use of MTBE-affected water, consistent with animal studies showing acute CNS depression from MTBE exposure. The current research evaluates acute CNS effects during bathing/showering by application of physiologically-based pharmacokinetic (PBPK) techniques to compare internal doses in animal toxicity studies to human exposure scenarios. An additional reference point was the delivered dose associated with the acute Minimum Risk Level (MRL) for MTBE established by the Agency for Toxic Substances and Disease Registry. A PBPK model for MTBE and its principal metabolite, t-butyl alcohol (TBA) was developed and validated against published data in rats and humans. PBPK analysis of animal studies showed that acute CNS toxicity after MTBE exposure can be attributed principally to the parent compound since the metabolite (TBA) internal dose was below that needed for CNS effects. The PBPK model was combined with an exposure model for bathing and showering which integrates inhalation and dermal exposures. This modeling indicated that bathing or showering in water containing MTBE at 1 mg/L would produce brain concentrations approximately 1000-fold below the animal effects level and twofold below brain concentrations associated with the acute MRL. These findings indicate that MTBE water concentrations of 1 mg/L or below are unlikely to trigger acute CNS effects during bathing and showering. However, MTBE's strong odor may be a secondary but deciding factor regarding the suitability of such water for domestic uses.

Comparison of contact site cancer potency across dose routes: case study with epichlorohydrin.

Risk assessment for airborne carcinogens is often limited by a lack of inhalation bioassay data. While extrapolation from oral-based cancer potency factors may be possible for some agents, this is not considered feasible for contact site carcinogens. The change in contact sites (oral: g.i. tract; inhalation: respiratory tract) when switching dose routes leads to possible differences in tissue sensitivity as well as chemical delivery. This research evaluates the feasibility to extrapolate across dose routes for a contact site carcinogen through a case study with epichlorohydrin (EPI). EPI cancer potency at contact sites is compared across three bioassays involving different dose routes (gavage, drinking water, inhalation) through the use of dosimetry models to adjust for EPI delivery to contact sites. Results indicate a large disparity (two orders of magnitude) in potency across the three routes of administration when expressed as the externally applied dose. However, when expressed as peak delivered dose, inhalation and oral potency estimates are similar and overall, the three potency estimates are within a factor of seven. The results suggest that contact site response to EPI is more dependent upon the rate than the route of delivery, with peak concentration the best way to extrapolate across dose routes. These results cannot be projected to other carcinogens without further study.

The arylation of microsomal membrane proteins by acetaminophen is associated with the release of a 44 kDa acetaminophen-binding mouse liver protein complex into the cytosol.

When analyzed by Western blotting with affinity purified antibodies against acetaminophen, proteins of molecular weight 44 and 58 kDa appear to be the major macromolecular targets in livers of mice administered hepatotoxic concentrations of acetaminophen. In this study, we have examined the characteristics and biochemical properties of the 44 kDa acetaminophen-binding protein in mouse liver. Data are presented which indicate that the 44-kDa protein is the earliest detectable protein targeted by acetaminophen; 30 min after acetaminophen administration in vivo, the binding to the 44 kDa protein is primarily localized in the microsomal fraction. After 1 hr, the 44 kDa acetaminophen-binding protein can be detected in both the microsomes and the cytosol. Extractions of microsomes with Triton X-114 or 1 M NaCl suggests that the acetaminophen-bound 44-kDa protein behaves as a peripheral membrane protein associated with the endoplasmic reticulum by ionic interactions. The cytosolic and microsomal 44-kDa proteins possess similar biochemical properties; both exist natively as components of a protein complex of greater than 200 kDa and both consist of two major isovariants with isoelectric points of 7.0 and 7.1 on two-dimensional gels. When N-acetyl-p-benzoquinone imine, the reactive metabolite of acetaminophen, is incubated with cytosolic or microsomal fractions from control liver, targeting of a 44-kDa protein is only observed in the microsomes. However, when acetaminophen is activated in an NADPH-regenerating microsomal system in vitro, some of the microsomal 44-kDa protein complex can be solubilized and released into the cytosol. Thus, acetaminophen administration can alter the subcellular distribution of at least one protein target in the cell.

DNA adduct formation in mouse tissues in relation to serum levels of benzo(a)pyrene-diol-epoxide after injection of benzo(a)pyrene or the diol-epoxide.

Previous studies have shown that the carcinogenic metabolite of benzo(a)pyrene [B(a)P], B(a)P-7,8-diol-9,10-epoxide (BPDE), is transported in serum after B(a)P injection in mice. It is possible that serum transport is an important source of carcinogenic metabolite and results in DNA adduct formation in tissues. This possibility was studied by comparing the time course for BPDE appearance in serum with that for BPDE/DNA adduct formation after B(a)P i.p. injection (2, 20, or 200 mg/kg) into female C57BL/6 x C3H F1 mice. Additionally, BPDE was injected i.v. (8.25 nmol), and its disappearance from serum and adduction of tissue DNA were followed. BPDE serum levels and DNA adduct levels were measured by 32P-postlabeling analysis. Results indicate that, after a 200-mg B(a)P/kg i.p. injection, BPDE/DNA adduct levels rose sharply in liver, lung, kidney, stomach, and spleen through 5 h and then more gradually through 24 h. Adduct levels were similar in all tissues at 24 h. BPDE levels in serum reached a plateau within 2.5 h and remained constant thereafter (10 to 11 nM). B(a)P levels in serum fell steadily from 1980 nM at 1 h to 350 nM by 24 h. Levels of serum BPDE and DNA adducts showed a similar dose dependency at 10- and 100-fold lower B(a)P i.p. doses. After BPDE i.v. injection, BPDE levels in serum decreased to 0.16% of the initial level within 5 min. By this time, BPDE/DNA adducts were at peak levels in all tissues assayed. Lung adduct levels were 10 to 100 times greater than those in the other tissues. These results support a role for serum transport of BPDE in the production of DNA adducts after B(a)P since BPDE was available in serum throughout the time course for DNA adduct formation. Further, injected BPDE rapidly formed DNA adducts and this occurred primarily in the lung, which had the greatest access to the transported carcinogen.

Transport of DNA-adducting metabolites in mouse serum following benzo[a]pyrene administration.

Metabolic activation of benzo[a]pyrene (BaP) by cellular enzymes is required for DNA adduct formation. In vivo DNA adducts might also arise from BaP metabolites supplied via the systemic circulation, rather than from in situ activation. We determined whether electrophilic metabolites could be detected in mouse serum 4 h after BaP dosing (i.p.) by trapping metabolites with salmon sperm DNA (ssDNA), followed by 32P-postlabeling analysis for DNA adducts. In vitro studies demonstrated that mouse serum sequesters BaP-7,8-diol-9,10-epoxide (BPDE) and protects it from hydrolysis. BPDE was rapidly transferred from serum to ssDNA or splenocytes, with adduct levels in ssDNA 4- to 7-fold greater than in splenocytes. After BaP administration, mouse serum produced two adduct spots when incubated with ssDNA. The major adduct (spot 3) co-chromatographed with a BPDE adduct standard, while the minor adduct (spot 2) was unrelated to BPDE. A BPDE standard curve in control serum was developed to quantitate BPDE levels in dosed serum. These levels ranged from 13.1 to 19.1 nM. Tissue DNA contained three adduct spots: spots 2 and 3 appeared identical to the respective adducts arising from dosed serum. BPDE-DNA adducts in tissues were highest in liver, lung and spleen, with kidney and stomach levels significantly lower. Levels of adduct 2 did not correlate with levels of adduct 3, especially in spleen where the adduct 2/adduct 3 ratio was very low. In vitro studies in which splenocytes were presented with both adducting metabolites suggested that splenocytes preferentially form adduct 3. These results indicate that two of the three BaP electrophilic metabolites responsible for cellular DNA damage are present in mouse serum. The levels of BPDE in serum may be sufficient to account for a substantial portion of the tissue load of BPDE-DNA adducts.

Benzo[a]pyrene-induced immunotoxicity: comparison to DNA adduct formation in vivo, in cultured splenocytes, and in microsomal systems.

Benzo[a]pyrene (BaP)/DNA adduct formation appears to be involved in carcinogenesis, but the relationship between adduct formation and BaP-induced immunotoxicity is unknown. We compared DNA adduct formation (32P-postlabeling analysis) to suppression of polyclonal immune responses (3H-TdR incorporation and IgM secretion) and decreases in cell viability in B6C3F1 female mouse splenic leukocytes (SPL). BaP administration (200 mg/kg, ip) resulted in suppression of polyclonal responses and substantial DNA adduct formation in mouse SPL. SPL adduct levels were similar to those in liver, lung, kidney, and stomach. In vitro exposure of SPL to BaP without rat liver activation enzymes (S9) caused decreases in SPL viability and immune responses that were dependent on dose and exposure period. However, DNA adduct formation in SPL was very low between 1 and 200 microM BaP. S9 enhanced the toxicity of BaP for SPL cultures. Adduct formation was rapid and dose related in +S9 incubates. The low level of BaP activation by SPL was confirmed in microsomal incubations in which splenic microsomes exhibited much lower aryl hydrocarbon hydroxylase (AAH) activity and ability to form DNA-adducting metabolites than did microsomes from liver or lung. Results indicate that immunosuppression produced by BaP in these systems was due to cytotoxic effects. It appears that these effects were caused by two separate mechanisms, one dependent on and one independent of DNA adduct formation. Since SPL had high levels of DNA adducts after ip injection of BaP, reactive metabolites of BaP may be involved in the immunotoxicity seen in vivo.

Ultrastructural changes during acute acetaminophen-induced hepatotoxicity in the mouse: a time and dose study.

This study was undertaken to evaluate the early ultrastructural changes during the development of acetaminophen hepatotoxicity. Doses at or near the threshold for hepatotoxicity were selected to permit comparison of early reversible effects to those which ultimately progressed to necrosis in the absence of early agonal effects or drug-induced mortality. Both 300- and 600-mg/kg doses resulted in similar declines in hepatic glutathione levels to 14 and 22% of control values, respectively, by 2 hours, with more rapid recovery after the low dose. Plasma sorbitol dehydrogenase activity was elevated after 600 mg/kg but not after 300 mg/kg. During the first 2 hours after acetaminophen there was cytomegaly with rapid progression to necrosis after 600 mg/kg but minimal progression after 300 mg/kg. Ultrastructurally, vesiculation, vacuolation and mitochondrial and plasma membrane degeneration culminated in scattered single cell death by 4 hours and widespread centrilobular necrosis by 8 hours after 600 mg/kg. The time course of lesion development was slower after 300 mg/kg with damage restricted to the first two to three rows of centrilobular cells and limited numbers of isolated necrotic cells by 8 hours. By 18 to 24 hours livers of mice given 300 mg/kg appeared normal. Results are consistent with the endoplasmic reticulum being the site of acetaminophen activation and initial attack. However, early ultrastructural changes in mitochondria and plasma membrane observed after the high dose were not prominent after the low dose. This suggests that early acetaminophen damage to these organelles may play a critical role in acetaminophen hepatotoxicity.