Derivation of no significant risk levels for three lower acrylates: Conclusions and recommendations from an expert panel.

A panel of toxicology, mode of action (MOA), and cancer risk assessment experts was engaged to derive no-significant-risk-levels (NSRLs) for three lower acrylates: methyl acrylate (MA), ethyl acrylate (EA), and 2-ethylhexyl acrylate (2EHA) using the best available science, data, and methods. The review was structured as a five-round, modified Delphi format, a systematic process for collecting independent and deliberative input from panel members, and it included several procedural elements to reduce potential sources of bias and groupthink. Input from the panel for key decisions in the dose-response assessments resulted in NSRL values of 530 µg/day (330-800 µg/day), 640 µg/day (280-670 µg/day), and 1700 µg/day (1300-2700 µg/day) for MA, EA, and 2EHA, respectively. Novel to this approach were the use of nonneoplastic lesions reported at point of contact where tumors have been reported in laboratory rodents, along with nonlinear extrapolation to low doses (uncertainty factor approach) based upon panel recommendations. Confidence in these values is considered medium to high for exposures applied to the routes of exposure tested (inhalation for MA and EA, dermal for 2EHA), but confidence is considered lower when applied to other routes of exposure.

Cancer weight of evidence for three lower acrylates: Conclusions and recommendations from an expert panel.

An international panel of experts was engaged to assess the cancer weight of evidence (WOE) for three lower acrylates: methyl acrylate, ethyl acrylate, and 2-ethylhexyl acrylate. The review was structured as a three-round, modified Delphi format, a systematic process for collecting independent and deliberative input from panel members, and it included procedural elements to reduce bias and groupthink. Based upon the available science, the panel concluded: (1) The MOA for point of contact tumors observed in rodent cancer bioassays that is best supported by available data involves increased cell replication by cytotoxicity and regenerative proliferation; (2) The WOE supports a cancer classification of "Not likely to be carcinogenic to humans" a conclusion that is more in line with an IARC classification of Group 3 rather than Group 2 B; (3) Quantitative cancer potency values based on rodent tumor data are not required for these chemicals; and (4) Human health risk assessment for these chemicals should instead rely on non-cancer, precursor endpoints observed at the point of contact (e.g., hyperplasia). The degree of consensus (consensus scores of 0.84-0.91 out of a maximum score of 1) and degree of confidence (7.7-8.7 out of a maximum score of 10) in the WOE conclusions is considered high.

Building scientific confidence in the development and evaluation of read-across.

Read-across is an alternative approach exploited to address information requirements for risk assessment and for regulatory programmes such as the European Union's REACH regulation. Whilst read-across approaches are accepted in principle, difficulties still remain in applying them consistently in practice. Recent work within Cefic LRI and ECETOC attempted to summarize the state-of-the-art and identify some of the barriers to broader acceptance of read-across approaches to overcome these. Acceptance is undoubtedly thwarted partly by the lack of a systematic framework to characterize the read-across justification and identify the uncertainties particularly for complex regulatory endpoints such as repeated-dose toxicity or prenatal developmental toxicity. Efforts are underway by the European Chemical's Agency (ECHA) to develop a Read-Across Assessment Framework (RAAF) and private sector experts have also considered the development of a similar framework. At the same time, mechanistic chemical categories are being proposed which are underpinned by Adverse Outcome Pathways (AOPs). Currently such frameworks are only focusing on discrete organic substances, though the AOP approach could conceivably be applied to evaluate more complex substances such as mixtures. Here we summarize the deliberations of the Cefic LRI read-across team in characterizing scientific confidence in the development and evaluation of read-across.

Coupling between MRI-assessed regional aortic pulse wave velocity and diameters in patients with thoracic aortic aneurysm: a feasibility study.

AIMS: Thoracic aortic aneurysm (TAA) is potentially life-threatening and requires close follow-up to prevent aortic dissection. Aortic stiffness and size are considered to be coupled. Regional aortic stiffness in patients with TAA is unknown. We aimed to evaluate coupling between regional pulse wave velocity (PWV), a marker of vascular stiffness, and aortic diameter in TAA patients. METHODS: In 40 TAA patients (59 ± 13 years, 28 male), regional aortic diameters and regional PWV were assessed by 1.5 T MRI. The incidence of increased diameter and PWV were determined for five aortic segments (S1, ascending aorta; S2, aortic arch; S3, thoracic descending aorta; S4, suprarenal and S5, infrarenal abdominal aorta). In addition, coupling between regional PWV testing and aortic dilatation was evaluated and specificity and sensitivity were assessed. RESULTS: Aortic diameter was 44 ± 5 mm for the aortic root and 39 ± 5 mm for the ascending aorta. PWV was increased in 36 (19 %) aortic segments. Aortic diameter was increased in 28 (14 %) segments. Specificity of regional PWV testing for the prediction of increased regional diameter was ≥ 84 % in the descending thoracic to abdominal aorta and ≥ 68 % in the ascending aorta and aortic arch. CONCLUSION: Normal regional PWV is related to absence of increased diameter, with high specificity in the descending thoracic to abdominal aorta and moderate results in the ascending aorta and aortic arch.

Ascending aorta curvature and flow displacement are associated with accelerated aortic growth at long-term follow-up: A MRI study in Marfan and thoracic aortic aneurysm patients.

BACKGROUND: Aortic aneurysm formation is associated with increased risk of aortic dissection. Current diagnostic strategies are focused on diameter growth, the predictive value of aortic morphology and function remains underinvestigated. We aimed to assess the long-term prognostic value of ascending aorta (AA) curvature radius, regional pulse wave velocity (PWV) and flow displacement (FD) on aortic dilatation/elongation and evaluated adverse outcomes (proximal aortic surgery, dissection/rupture, death) in Marfan and non-syndromic thoracic aortic aneurysm (NTAA) patients. METHODS: Long-term magnetic resonance imaging (MRI) and clinical follow-up of two previous studies consisting of 21 Marfan and 40 NTAA patients were collected. Baseline regional PWV, AA curvature radius and normalized FD were assessed as well as diameter and length growth rate at follow-up. Multivariate linear regression was performed to evaluate whether baseline predictors were associated with aortic growth.=. RESULTS: Of the 61 patients, 49 patients were included with MRI follow-up (n = 44) and/or adverse aortic events (n = 7). Six had undergone aortic surgery, no dissection/rupture occurred and one patient died during follow-up. During 8.0 [7.3-10.7] years of follow-up, AA growth rate was 0.40 ± 0.31 mm/year. After correction for confounders, AA curvature radius (p = 0.01), but not FD or PWV, was a predictor of AA dilatation. Only FD was associated with AA elongation (p = 0.01). CONCLUSION: In Marfan and non-syndromic thoracic aortic aneurysm patients, ascending aorta curvature radius and flow displacement are associated with accelerated aortic growth at long-term follow-up. These markers may aid in the risk stratification of ascending aorta elongation and aneurysm formation.

Biomonitoring as a tool in the human health risk characterization of dermal exposure.

Dermal exposure is an important factor in risk characterization. In occupational settings it becomes relatively more important because of the continuous reduction in inhalation exposure. In the public health arena, dermal exposure may also form a significant contribution to the total exposure. Dermal exposure, however, is difficult to assess directly because it is determined by a host of factors, which are difficult to quantify. As a consequence, dermal exposure is often estimated by application of models for external exposure. In combination with modeled or measured data for percutaneous penetration, these provide an estimate for the internal exposure that is directly related to the systemic effects. The advantages and drawbacks of EASE (Estimation and Assessment of Substance Exposure) and RISKOFDERM (Risk Assessment of Occupational Dermal Exposure), two models for external exposure that are mentioned in the Technical Guidance Document for the European Union risk assessments performed under the Existing Substances Regulation (EEC/793/93), are discussed. Although new chemicals regulation (REACh, 1907/2006/EC) is now in place in the European Union, the principles applied under the previous legislation do not change and the same models will continue to be used. The results obtained with these models for styrene, 2-butoxyethanol, and 1-methoxy-2-propanol in specific exposure scenarios are compared with an alternative method that uses biomonitoring data to assess dermal exposure. Actual external exposure measurements combined with measured or modeled percutaneous penetration data give acceptable results in risk assessment of dermal exposure, but modeled data of external dermal exposure should only be used if no other data are available. However, if available, biomonitoring should be considered the method of choice to assess (dermal) exposure.

The role of metallothionein in the reduction of cisplatin-induced nephrotoxicity by Bi3(+)-pretreatment in the rat in vivo and in vitro. Are antioxidant properties of metallothionein more relevant than platinum binding?

Nephrotoxicity induced by cisplatin (CDDP) was reported to be reduced by Bi3(+)-pretreatment, which selectively induces renal metallothionein (MT). In the present study renal MT had increased to 250% of control in rats that received bismuth subnitrate (50 mumol/kg/day, orally) for 8 days. In vitro experiments demonstrated that the reduction of CDDP-induced toxicity is a renal effect: in proximal tubular cells (PTC) isolated from Bi3(+)-treated rats the toxicity of CDDP, and also of HgCl2, CdCl2 and p-aminophenol, was reduced as compared to PTC from untreated rats. In contrast to the reduction in CDDP, Hg2+ and Cd2+ toxicity, the reduction in p-aminophenol toxicity cannot be explained by the metal-binding properties of MT. MT was reported to act as a free radical scavenger, which may explain our observation since p-aminophenol toxicity is thought to be a consequence of the generation of oxygen radicals. In vivo experiments showed that the overall renal Pt-content as well as the Pt bound to renal MT is lower in Bi3(+)-pretreated rats than in untreated rats, 24 hr after administration of CDDP (12 mg/kg), suggesting that the reduction in nephrotoxicity is not due to increased binding of Pt2+ to renal MT. Renal superoxide dismutase (SOD) activity was increased in rats that had only received CDDP. Such a rise in SOD may result from peroxidative damage caused by exposure to CDDP. The fact that SOD was not elevated in rats that received Bi3+ prior to CDDP suggests that (i) peroxidation contributes to CDDP-induced nephrotoxicity and (ii) the anti-oxidant properties of MT are responsible for the reduction of this toxicity.

Toxicity of the cysteine-S-conjugates and mercapturic acids of four structurally related difluoroethylenes in isolated proximal tubular cells from rat kidney. Uptake of the conjugates and activation to toxic metabolites.

Isolated proximal tubular cells from rat kidney were incubated with the cysteine-S-conjugates and corresponding mercapturates of the potent nephrotoxicants tetrafluoroethylene (TFE), chlorotrifluoroethylene (CTFE), 1,1-dichloro-2,2-difluoroethylene (DCDFE) and 1,1-dibromo-2,2-difluoroethylene (DBDFE). Toxicity of these S-conjugates was determined by their ability to inhibit alpha-methylglucose uptake by the cells. The cytotoxicity of the cysteine-S-conjugates and mercapturates of TFE and CTFE was similar, but the cysteine-S-conjugates of DCDFE and DBDFE were more toxic than their mercapturates. The cytotoxicity of the conjugates decreased in the following order TFE approximately CTFE greater than DCDFE greater than DBDFE, which is the same as observed in vivo. Inhibition of renal cysteine-S-conjugate beta-lyase by aminooxyacetic acid alleviated the cytotoxicity of both the cysteine-S-conjugates and the mercapturic acids of the four haloethylenes. The cytotoxicity of the mercapturates, but not of the cysteine-S-conjugates, could be reduced by probenecid, suggesting that the cysteine-S-conjugates are transported by a different carrier system than the mercapturates. The deacetylation of the mercapturates of TFE and CTFE in the cells was much higher than that of the mercapturates of DCDFE and DBDFE. The cysteine-S-conjugates of DCDFE and DBDFE were N-acetylated by the cells whereas the other cysteine-S-conjugates were not (TFE) or only marginally (CTFE) N-acetylated. The observed differences in cytotoxicity may be explained by differences in (1) the balance between acetylation/deacetylation by the cells, (2) the conversion rate of the S-conjugates to toxic metabolites by renal beta-lyase and (3) the transport into the proximal tubular cells.

4-Methylthiobenzoic acid reduces cisplatin nephrotoxicity in rats without compromising anti-tumour activity.

Administration of 4-methylthiobenzoic acid (MTBA) (100 mg/kg) strongly reduced cisplatin nephrotoxicity (7.5 mg/kg, 20 min after MTBA) in rats as determined by histopathology and blood urea nitrogen. Anti-tumour activity against a colonic adenocarcinoma, CC 531, that was implanted in rats, was unaffected by MTBA pretreatment. Studies with isolated renal proximal tubular cells (PTC) demonstrated that preincubation of the cells with MTBA diminished cisplatin nephrotoxicity in vitro as it did in vivo. Preincubation of the PTC with probenecid completely abolished the protective effect of MTBA against cisplatin toxicity. These data indicate that MTBA is actively transported into the PTC. The mechanism of action of MTBA was investigated by NMR studies which showed that cisplatin and cis-diamminediaquaplatinum(II), its hydrolysis product, reacted with the methylthio-sulphur. We suggest that MTBA after selective accumulation in the kidney inactivates cisplatin intracellularly by nucleophilic attack of the methylthio-sulphur to the Pt-moiety. Since MTBA shows no acute toxicity in the rat, even if administered at very high doses, it may be useful to suppress the nephrotoxic side effects of cisplatin anti-tumour therapy.

Primary culture of proximal tubular cells from normal rat kidney as an in vitro model to study mechanisms of nephrotoxicity. Toxicity of nephrotoxicants at low concentrations during prolonged exposure.

The aim of this study was to set up an in vitro system to study nephrotoxicity of xenobiotics which allows exposure at low concentrations for long periods (1-5 days). A very pure preparation of isolated proximal tubular cells (PTC) from rat kidney (Boogaard et al., Toxicol Appl Pharmacol 101: 135-143, 1989) was brought into primary culture. Cells grew to confluence in 3 days and could be maintained up to 8 days in a modification of Dulbecco's modified Eagle's medium Ham F12 nutrient mixture supplemented with fetal calf serum. Fibroblast growth was completely suppressed by replacement of L-valine by D-valine and of L-arginine by L-ornithine. Polarity was retained: in cells grown on filters organic anions were transported at the basolateral membrane while D-glucose transport was located at the apical membrane. Inhibition of the latter was used to assess the functional integrity of the cells after exposure to nephrotoxins. The newly grown cells expressed gamma-glutamyltranspeptidase activity since incubation with the glutathione-conjugate of 1,1-dichloro-2,2-difluoroethylene (DCDFE) induced cytotoxicity. Both beta-lyase and acylase activities were expressed because the cysteine-S-conjugate and the corresponding mercapturate of DCDFE showed cytotoxicity. Cultured cells showed toxicity on prolonged exposure to very low concentrations of gentamicin, cephaloridine, cisplatin and the cysteine-S-conjugate of chlorotrifluoroethylene. The lowest concentrations at which toxicity can be observed are 1-3 orders of magnitude lower in primary cultures than in freshly isolated PTC in suspension. This indicates that this cell model is suitable to investigate mechanisms of nephrotoxicity in vitro, at prolonged exposure to the low concentrations that are relevant in vivo levels.

Suitability of S-phenyl mercapturic acid and trans-trans-muconic acid as biomarkers for exposure to low concentrations of benzene.

Phenol is not reliable as a biomarker for exposure to benzene at concentrations below 5 ppm (8-hr time-weighted average [TWA]). S-Phenylmercapturic acid (S-PMA) and trans-trans-muconic acid (tt-MA), two minor urinary metabolites of benzene, have been proposed as biomarkers for low-level exposures. The aim of this study was to compare their suitability as biomarkers. S-PMA and tt-MA were determined in 434 urine samples collected from 188 workers in various settings in the petrochemical industry and from 52 control workers with no occupational exposure to benzene. Benzene concentrations in the breathing zone of the potentially exposed workers were assessed by personal air monitoring. Strong correlations were found between S-PMA and tt-MA concentrations in end-of-shift samples and between either of these parameters and airborne benzene concentrations. Exposure to 1 ppm benzene (8-hr TWA) leads to an average concentration in end-of-shift samples of 21 mol S-PMA and 1.5 mmol tt-MA per mol creatinine. Of an inhaled dose of benzene, on average 0.11% (range 0.05-0.26%) was excreted as S-PMA with an apparent elimination half-life of 9.1 (standard error [SE] 0.7) hr and 3.9% (range 1.9-7.3%) as tt-MA with a half-life of 5.0 (SE 0.5) hr. Due to its longer elimination half-life, S-PMA proved a more reliable biomarker than tt-MA for benzene exposures during 12-hr shifts. Specificity of S-PMA, but not tt-MA, was sufficient to discriminate between the 14 moderate smokers and the 38 nonsmokers from the control group. The mean urinary S-PMA was 1.71 (SE 0.27) in smokers and 0.94 (SE 0.15) mol/mol creatinine in nonsmokers (p = 0.013). The mean urinary tt-MA was 0.046 (SE 0.010) in smokers and 0.029 (SE 0.013) mmol/mol creatinine in nonsmokers (p = 0.436). The inferior specificity of tt-MA was due to relatively high background values of up to 0.56 mmol/mol creatinine, which may be found in nonexposed individuals and limits the use of tt-MA to concentrations of benzene over 1 ppm (8-hr TWA). We conclude that S-PMA is superior to tt-MA as a biomarker for low-level benzene exposures because it is more specific, enabling reliable determination of benzene exposures down to 0.3 ppm (8-hr TWA), and because its longer half-life makes it more suited for biological monitoring of operators working in shifts longer than 8 hr.

Biological monitoring of exposure to 3-chloro-4-fluoroaniline by determination of a urinary metabolite and a hemoglobin adduct.

In two studies, involving 75 and 72 workers, potential exposure to 3-chloro-4-fluoroaniline (CFA) was biologically monitored by determination of its main urinary metabolite 2-amino-4-chloro-5-fluorophenol sulfate (CFA-S). As this method only allows the detection of recent exposure, analysis of CFA adducts bound to hemoglobin (Hb) was investigated as a method that allows biological monitoring of exposure to CFA over longer periods. The median CFA-S concentration in 67 samples from the first study was 0.14 mumole/g creatinine (range < 0.05-2.82) and in 201 samples from the second study 0.21 mumole/g creatinine (range < 0.05-6.05). In addition, urine samples, collected after shifts with supposed incidental exposure, slightly higher concentrations were measured: 0.27 mumole/g creatinine (range < 0.05-122; 18 samples) and 0.76 mumole/g creatinine (range < 0.05-18.5; 46 samples), respectively. The median Hb adduct concentration in 75 samples from the first study was 9 pmoles CFA/g Hb (range < 5-640) and in 46 samples from the second study 12 pmoles/g Hb (range 3-24). In 24 blood samples collected after incidents, a median concentration of 13 pmoles CFA/g Hb (range < 5-52) was found. Urinary CFA-S and Hb adducts correlated well in samples collected shortly after incidental exposures. However, in 25% of the operators, no CFA-S was detected during routine biological monitoring while Hb adduct analysis showed clear evidence of exposure. This indicates that because of the stability of Hb adducts of CFA in blood, intermittent exposure to CFA is more reliably biologically monitored by determination of Hb adducts of CFA than by assessment of urinary CFA-S.

Molecular dosimetry of 2,4-difluoroaniline in humans and rats by determination of hemoglobin adducts.

Exposure to 2,4-difluoroaniline (DFA) was monitored by GC-MS of DFA adducts bound to hemoglobin (Hb). In two studies, involving 20 and 16 workers potentially exposed to low concentrations of DFA, median concentrations of 10 (range 1-83) and 20 (range 4-322) pmole/g Hb were found, respectively. For better interpretation of these results, the in vivo binding of DFA to Hb was investigated. DFA was administered orally at doses of 0, 0.078, 0.775, 7.75, and 77.5 mumole/kg/day, to 10 male and 10 female Fischer 344 rats for 10 consecutive days (2 rats/sex/dose group). A linear relation between dose and adduct concentration was observed. At the two lowest doses (0.078 and 0.775 mumole/kg/day) no methemoglobinemia was observed, but adducts could easily be measured. At these doses, the mean adduct levels were in the same range as found in the human studies. As yet, no occupational exposure limit for DFA has been established. The German biological tolerance value (BAT-value) for aniline was set at 7.2 nmole/g Hb. This BAT-value is based on the relation between methemoglobinemia and adduct formation. The amount of Hb binding by aniline and DFA was found to be similar in the rat. Assuming that this is also the case in humans, the BAT-value for aniline may tentatively be used for DFA as well. In both studies of occupationally exposed workers, the adduct levels were well below this BAT-value.

Biomarkers of exposure to 1,3-butadiene as a basis for cancer risk assessment.

1,3-Butadiene (BD) is carcinogenic in mice and rats, with mice being considerably more sensitive than rats. Urine metabolites are 1, 2-dihydroxybutyl mercapturic acid (DHBMA) and a mixture of monohydroxy-3-butenyl mercapturic acids (MHBMA). The reactive metabolite 1,2-epoxy-3-butene forms 1- and 2-hydroxy-3-butenyl valine adducts in hemoglobin (MHBVal). The objectives of the study were (1) to compare the suitability of MHBMA, DHBMA, and MHBVal as biomarkers for low levels of exposure to BD, and (2) to explore relative pathways of metabolism of BD in humans for comparison with mice and rats, which is important in relation to cancer risk assessment in man. Analytical methods of measuring MHBMA, DHBMA, and MHBVal were modified and applied in 2 studies to workers engaged in the manufacture and use of BD. Airborne BD concentrations were assessed by personal air monitoring. MHBMA in urine was more sensitive for monitoring recent exposures to BD when compared to DHBMA and could measure 8-h time weighted average exposures as low as 0.13 ppm. Relatively high natural background levels in urine restricted the sensitivity of DHBMA. The origin of this background is currently unknown. The measurement of MHBVal adducts in hemoglobin was a sensitive method for monitoring cumulative exposures to BD at or above 0.35 ppm. Statistically significant relationships were found between urinary MHBMA and DHBMA concentrations, between either of these variables and 8-h airborne BD levels and between MHBVal adducts and average airborne BD levels over 60 days. The data on biomarkers demonstrated a much higher rate of hydrolytic metabolism of 1,2-epoxy-3-butene in humans compared to mice and rats, which was reflected in a much higher DHBMA/(DHBMA + MHBMA) ratio and in much lower levels of MHBVal in humans. Assuming a genotoxic mechanism, the data of this study, coupled with other published data on DNA and hemoglobin binding in mice and rats, suggest that the cancer risk for man from exposure to BD is expected to be less than for the rat and much less than for the mouse.

Disposition of [Ring-U-(14)C]styrene in rats and mice exposed by recirculating nose-only inhalation.

The disposition of styrene was studied in a group of 12 Sprague Dawley rats and two groups of 30 CD1 mice exposed separately to 160 ppm [ring-U-(14)C]styrene of high specific radioactivity of 1.92 TBq x mol(-1) (52 Ci x mol(-1)) for 6 h. A nose-only exposure system was successfully adapted to (1) recirculate a portion of the flow to limit the amount of (14)C-styrene required, and (2) avoid any polymerization of the compound. The mean uptake of styrene in rats was 113 +/- 7 micromol x kg(-1) x h(-1) and stable over time. The mean uptake in mice was higher, 189 +/- 53 and 183 +/- 76 micromol x kg(-1) x h(-1), for the first and second mouse inhalation experiment, but decreased steadily over time. Some of the mice, but none of the rats, showed signs of overt toxicity. The overall excretion of styrene and its metabolites was quantitatively similar in rats and mice. Urinary excretion was the primary route of excretion while fecal excretion accounted for only a very small part of the radioactivity. There was, however, a significant difference between mice and rats in the exhalation of (14)CO(2), which must have resulted from opening and subsequent breakdown of the aromatic ring. In mice the exhalation of (14)CO(2) accounted for 6.4 +/- 1.0 and 8. 0 +/- 0.5% of the styrene retained during the first and second mouse inhalation experiment. In rats, exhalation of (14)CO(2) accounted for only 2.0 +/- 0.7% of the retained styrene. Together with the results from the quantitative whole-body autoradiography (showing significantly higher binding in mouse lung and nasal passages compared to rat) the larger production of (14)CO(2) might be indicative of the formation of reactive ring-opened metabolites in the mouse lung, which, in turn, might be related to the observed development of bronchioalveolar tumors and nasal effects in mice exposed to styrene.

Quantification of DNA adducts formed in liver, lungs, and isolated lung cells of rats and mice exposed to (14)C-styrene by nose-only inhalation.

Bronchiolo-alveolar tumors were observed in mice exposed chronically to 160 ppm styrene, whereas no tumors were seen in rats up to concentrations of 1000 ppm. Clara cells, which are predominant in the bronchiolo-alveolar region in mouse lungs but less numerous in rat and human lung, contain various cytochrome P450s, which may oxidize styrene to the rodent carcinogen styrene-7,8-oxide (SO) and other reactive metabolites. Reactive metabolites may form specific DNA adducts and induce the tumors observed in mice. To determine DNA adducts in specific tissues and cell types, rats and mice were exposed to 160 ppm [ring-U-(14)C]styrene by nose-only inhalation for 6 h in a recirculating exposure system. Liver and lungs were isolated 0 and 42 h after exposure. Fractions enriched in Type II cells and Clara cells were isolated from rat and mouse lung, respectively. DNA adduct profiles differed quantitatively and qualitatively in liver, total lung, and enriched lung cell fractions. At 0 and 42 h after exposure, the two isomeric N:7-guanine adducts of SO (measured together, HPEG) were present in liver at 3.0 +/- 0.2 and 1.9 +/- 0.3 (rat) and 1.2 +/- 0.2 and 3.2 +/- 0.5 (mouse) per 10(8) bases. Several other, unidentified adducts were present at two to three times higher concentrations in mouse, but not in rat liver. In both rat and mouse lung, HPEG was the major adduct at approximately 1 per 10(8) bases at 0 h, and these levels halved at 42 h. In both rat Type II and non-Type II cells, HPEG was the major adduct and was about three times higher in Type II cells than in total lung. For mice, DNA adduct levels in Clara cells and non-Clara cells were similar to total lung. The hepatic covalent binding index (CBI) at 0 and 42 h was 0.19 +/- 0.06 and 0.14 +/- 0.03 (rat) and 0. 25 +/- 0.11 and 0.44 +/- 0.23 (mouse), respectively. The pulmonary CBIs, based on tissues combined for 0 and 42 h, were 0.17 +/- 0.04 (rat) and 0.24 +/- 0.04 (mouse). Compared with CBIs for other genotoxicants, these values indicate that styrene has only very weak adduct-forming potency. The overall results of this study indicate that DNA adduct formation does not play an important role in styrene tumorigenicity in chronically exposed mice.

Quantitative and qualitative differences in the metabolism of 14C-1,3-butadiene in rats and mice: relevance to cancer susceptibility.

1,3-Butadiene (butadiene) is a potent carcinogen in mice, but not in rats. Metabolic studies may provide an explanation of these species differences and their relevance to humans. Male Sprague-Dawley rats and B6C3F1 mice were exposed for 6 h to 200 ppm [2,3-14C]-butadiene (specific radioactivity [sa] 20 mCi/mmol) in a Cannon nose-only system. Radioactivity in urine, feces, exhaled volatiles and 14C-CO2 were measured during and up to 42 h after exposure. The total uptake of butadiene by rats and mice under these experimental conditions was 0.19 and 0.38 mmol (equivalent to 3.8 and 7.5 mCi) per kg body weight, respectively. In the rat, 40% of the recovered radioactivity was exhaled as 14C-CO2, 70% of which was trapped during the 6-h exposure period. In contrast, only 6% was exhaled as 14C-CO2 by mice, 3% during the 6-h exposure and 97% in the 42 h following cessation of exposure. The formation of 14C-CO2 from [2,3-14C]-labeled butadiene indicated a ready biodegradability of butadiene. Radioactivity excreted in urine accounted for 42% of the recovered radioactivity from rats and 71% from mice. Small amounts of radioactivity were recovered in feces, exhaled volatiles and carcasses. Although there was a large measure of commonality, the exposure to butadiene also led to the formation of different metabolites in rats and mice. These metabolites were not found after administration of [4-14C]-1,2-epoxy-3-butene to animals by i.p. injection. The results show that the species differences in the metabolism of butadiene are not simply confined to the quantitative formation of epoxides, but also reflect a species-dependent selection of metabolic pathways. No metabolites other than those formed via an epoxide intermediate were identified in the urine of rats or mice after exposure to 14C-butadiene. These findings may have relevance for the prediction of butadiene toxicity and provide a basis for a revision of the existing physiologically based pharmacokinetic models.

Hepatic and pulmonary glutathione conjugation of 1,2:3,4-diepoxybutane in human, rat, and mouse in vitro.

1,3-Butadiene (BD) is a carcinogen in rats and mice. Previous in vitro studies showed that mouse liver microsomes formed 1,2-epoxy-3-butene (BMO) from BD and 1,2:3,4-diepoxybutane (BDE) from BMO at much higher rates than rat or human microsomes. Blood and tissue levels of BDE were significantly lower in rats than in mice following exposure to BD. Since mice are much more susceptible to cancer induced by BD than rats, these findings suggest a key role for BDE in BD-induced carcinogenicity. The aim of this study was to characterize the glutathione (GSH) conjugation of BDE by cytosol from human liver and mouse and rat liver and lung in vitro. BDE and radiolabeled GSH were incubated with cytosol. Conjugates were identified by 13C-NMR and FAB mass spectroscopy and quantitated by HPLC. The enzyme kinetics for the conjugation of BDE with GSH suggest that the higher BDE blood concentrations in mice compared with rats following inhalation exposure to BD are not due to differences in GSH conjugation of BDE.

Nephrotoxicity of halogenated alkenyl cysteine-S-conjugates.

In 1916 a relationship was postulated between the occurrence of aplastic anaemia in cattle and the soy bean meal that they had been fed, which had been extracted with trichloroethylene. The toxic compound was later identified as S-(1,2-dichlorovinyl)-L-cysteine (DCV-Cys). In addition to effects on the hemopoietic system it also produced nephrotoxicity in calves. In rats only renal tubular necrosis was found. Further research demonstrated that other halogenated hydrocarbons produced similar nephrotoxicity. The haloalkenyl cysteine-S-conjugates (Cys-S-conjugates) have extensively been studied; this has provided new insight into the biochemical processes that lead to nephrotoxicity. It has been shown that a combination of transport processes and specific metabolic pathways, resulting in reactive intermediates that bind to cellular macromolecules, makes the kidney vulnerable to the noxious effects of the haloalkenyl Cys-S-conjugates. The first part of this review gives a brief overview of the bioactivation of the haloalkenes; in the second part the present knowledge of the underlying mechanisms of cytotoxicity will be outlined.

Regeneration experiments of the platinated enzyme fumarase, using sodium diethyldithiocarbamate, thiourea, and sodium thiosulfate.

The enzyme fumarase is inhibited by [cis-Pt(NH3)2(H2O)2] (NO3)2. The Pt compound most likely binds at a S-methionine site. Sodium diethyldithiocarbamate (Naddtc) appears to be a powerful regenerator of enzymatic activity. Thiourea is less active, while sodium thiosulfate (STS) is almost inactive in restoring the activity of the enzyme. The regeneration phenomena are based on the dissociation of the Pt-S bonds of the methionine type, and formation of species like [Pt(ddtc)2]. In the model adduct [Pt(dien)GS-Me]2+ Naddtc, thiourea and STS easily break the Pt-S bond of the methionine type. It is concluded that the model system for Naddtc and thiourea does resemble fumarase quite well. S-donor ligands, which may be used as rescue agents in Pt antitumor therapy, are known to suppress nephrotoxicity caused by [cis-PtCl2(NH3)2]. A parallel is drawn between the enzyme reactivation, modeled by fumarase, and the [cis-PtCl2(NH3)2] nephrotoxicity suppression by rescue agents. It is proposed that a Pt-methionine type binding is broken by the rescue agents Naddtc and thiourea, but that the rescue agent STS only inhibits the nephrotoxicity by inactivating unbound Pt species in the cell.