Abstract  Male B6C3F1 mice were exposed to 4000 ppm methylene chloride (MC) for 6 hr/day, 5 days/week for up to 13 weeks. Groups of mice were killed at intervals from Day 2 to Week 13. Whole lungs were examined morphologically, immunocytochemically, and biochemically. Biochemical and morphological examination was also performed on isolated Clara cells. The major initial morphological effect seen in lungs was acute Clara cell damage after one exposure to MC. However, this damage appeared to resolve after five consecutive daily exposures to MC. After a 2-day interval the Clara cell lesion reappeared on subsequent reexposure to MC. However, the severity of the lesion decreased over the duration of the study. The appearance and disappearance of the lesion in the Clara cell correlated well with the activity of cytochrome P450 monooxygenase in the Clara cell as assessed immunocytochemically (cytochromes P450IIB 1 and 2) in the whole lung and biochemically in the freshly isolated Clara cell (determined by ethoxycoumarin O-dealkylation and aldrin epoxidation). When there was a marked decrease in cytochrome P450 monooxygenase activity the lesion was not present. This suggested that with time the lung (Clara cell) has developed tolerance to MC possibly due to the inactivation of a cytochrome P450 isozyme. The glutathione S-transferase metabolism of MC by the lung cytosol remained virtually unaltered throughout the study. Events accompanying the discussed changes include (1) a significant increase in nonprotein sulfhydryl in the lungs of all exposed animals, (2) altered plating characteristics of the isolated Clara cells from exposed lungs after 24 hr in culture, and (3) an increase in the number of bronchiolar cells in the S-phase after the first exposure to MC. The study also demonstrates the advantages of target cell isolation and study over whole lung biochemical investigation alone.

Abstract  Bundesminister fur Forschung und Technologie.

Abstract  The reports of elevated carboxyhemoglobin (COHb) levels following exposure to dichloromethane (DCM) stimulated the research on the toxicology of this solvent. The metabolic pathway of DCM to carbon monoxide (CO) involves an oxygen insertion catalyzed by cytochrome P-450 (Gargas et al 1986), mainly by the isozyme cytochrome P-450 IIE1 (CYP2E1). Pyrazole (PYR) is known to depress the oxidative metabolism of DCM, but based upon the catalytic, spectral, and immunological properties it appears to induce CYP2E1 (Palakodety et al 1988). The effect of PYR on carboxyhemoglobinemia following DCM administration in rats was investigated.

Abstract  The purpose of this investigation was to determine whether similar carboxyhemoglobin (COHb) values obtained from inhaling carbon monoxide or methylene chloride have the same behavioral effects. The effects were assessed by the performance of 12 humans on a visual-manual, dual-task, and an auditory vigilance task. The results indicated that both substances in concentrations sufficient to produce 5-percent COHb significantly impaired human performance under difficult or demanding task conditions. The conclusion was that carbon monoxide, the main metabolite of methylene chloride, was responsible for the observed performance decrements.

Abstract  The public depends on competent risk assessment from the federal government and the scientific community to grapple with the threat of pollution. When risk reports turn out to be overblown--or when risks are overlooked--public skepticism abounds. This comprehensive and readable book explores how the U.S. Environmental Protection Agency (EPA) can improve its risk assessment practices, with a focus on implementation of the 1990 Clean Air Act Amendments. With a wealth of detailed information, pertinent examples, and revealing analysis, the volume explores the "default option" and other basic concepts. It offers two views of EPA operations: The first examines how EPA currently assesses exposure to hazardous air pollutants, evaluates the toxicity of a substance, and characterizes the risk to the public. The second, more holistic, view explores how EPA can improve in several critical areas of risk assessment by focusing on cross-cutting themes and incorporating more scientific judgment. This comprehensive volume will be important to the EPA and other agencies, risk managers, environmental advocates, scientists, faculty, students, and concerned individuals.

Abstract  In a recent report, we found an elevated risk of cancer of the central nervous system (CNS) in several occupations and industries, and a modest association with exposure to solvents and to contact with the public.

To further explore the occupational risk of CNS cancer among women, we extended the analysis of the previous death certificate-based case-control study, including 12,980 female cases (ICD-9 codes 191 and 192) in 24 US states in 1984-1992 and 51,920 female controls who died from diseases other than malignancies and neurological disorders. We applied newly designed job-exposure matrices for 11 occupational hazards, previously reported as brain cancer risk factors, to the occupation and industry codes in the death certificates. We also conducted a separate analysis of 161 meningioma cases (ICD-9 codes 192.1 and 192.3), a tumor more frequent among women, particularly in the postmenopausal age group.

Overall, CNS cancer risk showed a 20-30% increase among women exposed to electromagnetic fields (EMF), methylene chloride, insecticides and fungicides, and contact with the public. Risk for meningioma was elevated among women exposed to lead (OR = 1.9; 95% CI 1.0, 3.9). CNS cancer did not show a clear pattern of risk increase by probability and intensity of exposure to any of the explored risk factors. Cross-classification by probability and intensity of exposure did not reveal any significant trend. Cases were too few to explore trends of meningioma by probability and intensity of exposure to lead.

We did not find evidence of a strong contribution of 11 occupational hazards to the etiology of CNS cancer. However, limitations of the occupational information might have reduced our ability to detect clear patterns of risk.

Abstract  Methylene chloride has been the subject of recent toxicological and carcinogenesis studies because of significant human exposure and widespread use in industrial processing, food preparation and agriculture. In this study, liver and lung tumors, induced in female B6C3F1 mice by inhalation of 2000 p.p.m. methylene chloride (6 h/day, 5 days/week continuous exposure), were examined for the presence of activated ras proto-oncogenes. DNA was isolated from 49 spontaneous and 50 methylene chloride-induced liver tumors and screened by oligonucleotide hybridization of PCR amplified H-ras gene fragments for codon 61 mutations. In the chemically induced tumors, 38 mutations were detected, 16 C to A transversions in base 1, 16 A to G transitions in base 2 and 6 A to T transversions in base 2. This mutation profile was similar to that identified for the H-ras gene in the spontaneous liver tumors and suggests that methylene chloride acts in liver by promoting cells with spontaneous lesions. Tumors in which H-ras codon 61 mutations were not detected were examined for the presence of transforming genes by the nude mouse tumorigenicity assay. Except for activated K-ras genes detected in DNA from two methylene chloride induced tumors and one spontaneous tumor, no other transforming genes were identified. DNA from 54 lung tumors was screened by direct sequencing of PCR amplified DNA fragments of the K-ras gene for first and second exon mutations, and 12 mutations were identified, 5 in exon one and 7 in exon 2. The low number of spontaneous tumors available in this study limits the interpretation of the data, and thus the frequency and spectrum of K-ras activation in the methylene chloride induced tumors was not significantly different from that in the seven spontaneous tumors analyzed. Since K-ras activation was not detected in 80% of the tumors, the nude mouse tumorigenicity assay was used to examine the lung tumors for the presence of other transforming genes. At present no transforming genes other than ras genes were identified in either liver or lung tumors.

Abstract  The activity of cytochrome P450 (CYP) enzymes, which determine the rate of elimination of lipid-soluble drugs, demonstrates considerable interindividual variability. The extent to which age and sex influence CYP activity remains unclear in humans. Our objectives were to determine whether in vivo activity of selected CYP enzymes is affected by age or sex and to evaluate sex bioequivalence in a large sample size.

We have assessed in vivo activity of the CYP1A2, 2C19, 2D6, 2E1, and 3A4 enzymes in 161 normal subjects (51% female subjects and 40% aged >50 years). After simultaneous administration of a cocktail of selective probes (caffeine, mephenytoin, debrisoquin [INN, debrisoquine], chlorzoxazone, and dapsone, respectively), phenotypic indices for metabolism of these drugs were used as measures of individual CYP activity. Sex bioequivalence analysis used the bootstrap method.

There were no sex differences associated with CYP1A2 activity. A significant negative correlation (r = -0.572, P < .01) between enzyme activity and age was observed for CYP2C19, but there were no sex differences. CYP2D6 activity showed no dependence on age or sex. In contrast, CYP2E1 activity showed an age-associated increase (r = 0.393, P < .01), which developed earlier in life in male subjects compared with female subjects. These results were further supported by the sex bioequivalence analysis of CYP phenotypic activity, which revealed that sexes were equivalent with respect to CYP2C19 (90% confidence interval [CI], 0.874-1.04), CYP3A4 (90% CI, 0.95-1.176), and CYP2D6 (90% CI, 0.928-1.09) phenotype and just exceeded the 0.8 to 1.25 limits to be equivalent with respect to CYP2E1 (90% CI, 0.785-1.08) and CYP1A2 (90% CI, 0.736-1.03) phenotype.

These observations suggest that the presence of selective mechanisms of regulation for individual CYP enzymes can be influenced by age and sex. However, we suggest that sex has a limited ability to explain intersubject variation of activity for these phenotypic measures of CYP enzyme activity.

Abstract  This study examined the relationship between birthweight and exposure to emissions of methylene chloride (DCM) from manufacturing processes of the Eastman Kodak Company at Kodak Park in Rochester, Monroe County, New York. County census tracts were categorized as exposed to high, moderate, low or no DCM based on the Kodak Air Monitoring Program (KAMP) model, a theoretical dispersion model of DCM developed by Eastman Kodak Company. Birthweight and information on variables known to influence birthweight were obtained from 91,302 birth certificates of white singleton births to Monroe County residents from 1976 to 1987. No significant adverse effects of exposure to DCM on birthweight were found. Adjusted birthweight in high exposure census tracts was 18.7 g less than in areas with no exposure (95% confidence interval for the difference between high and no exposure - 51.6, 14.2 g). Problems inherent in the method of estimation of exposure, which may decrease power or bias the results, are discussed. Better methods to estimate exposure to emissions from multiple industrial point sources are needed.

Abstract  Dichloromethane dehalogenase from Methylophilus sp. DM11 is a glutathione S-transferase homolog that is specifically active with dihalomethane substrates. This bacterial enzyme and rat liver glutathione S-transferases were purified to investigate their relative reactivity with CH2Cl2 and related substrates. Rat liver alpha class glutathione transferases were inactive and mu class enzymes showed low activity (7-23 nmol/min/mg of protein) with CH2Cl2. theta class glutathione transferase 5-5 from rat liver and Methylophilus sp. dichloromethane dehalogenase showed specific activities of > or = 1 mumol/min/mg of protein. Apparent Kcat/Km were determined to be 3.3 x 10(4) and 6.0 x 10(4) L M-1 S-1 for the two enzymes, respectively. Dideutero-dichloromethane was processed to dideutereo-formaldehyde, consistent with a nucleophilic halide displacement mechanism. The possibility of a GSCH2X reaction intermediate (GS, glutathione; X, halide) was probed using CH2ClF to generate a more stable halomethylglutathione species (GSCH2F). The reaction of CH2ClF with dichloromethane dehalogenase produced a kinetically identifiable intermediate that decomposed to formaldehyde at a similar rate to synthetic HOCH2CH2SCH2F. 19F-NMR revealed the transient formation of an intermediate identified as GSCH2F by its chemical shift, its triplet resonance, and H-F coupling constant consistent with a fluoromethylthioether. Its decomposition was matched by a stoichiometric formation of fluoride. These studies indicated that the bacterial dichloromethane dehalogenase directs a nucleophilic attack of glutathione on CH2Cl2 to produce a halomethylthioether intermediate. This focuses attention on the mechanism used by theta class glutathione transferases to generate a halomethylthioeter from relatively unreactive dihalomethanes.

Abstract  Cytochrome P4502E1 (CYP2E1) is expressed in human peripheral blood lymphocytes (PBLs), and previous reports have suggested the possibility of using this readily available tissue as a reporter of CYP2E1 status. To further explore the relevance of this approach we assessed CYP2E1 expression in PBLs in two contrasting conditions, chronic hepatitis C and insulin-dependent diabetes (IDD), illustrating an organ and a systemic disease, respectively.

Total RNA was isolated from extracted PBLs (hepatitis C patients + IDD) and by percutaneous needle biopsy (hepatitis C patients only). Gene expression for CYP2E1 was determined by real-time reverse-transcription polymerase chain reaction. Histological changes in liver tissue were assessed according to Ludwig's criteria.

In patients with chronic hepatitis C a clear relationship was found between CYP2E1 expression in the liver and the progression of hepatic disease (both lobular inflammation and fibrosis indices), and observed variations were consistent with the preferential distribution of CYP2E1 in the lobular zone. No effect of the liver disease was, however, found at the PBL level. A statistically significant increase in mean CYP2E1 expression level was observed in the lymphocytes from poorly controlled IDD subjects compared to controls.

Taken together, our data indicate that the measurement of CYP2E1 expression in PBLs is not useful in liver diseases. However, in a systemic condition (IDD) this measurement can be proposed for monitoring the CYP2E1 induction in a relatively noninvasive manner. This tool should therefore be further validated in clinical field or experimental studies for CYP2E1 phenotyping purposes.

Abstract  Despite the very wide recognition that carbon monoxide (CO) is a significant neurotoxicant, the level at which subtle effects occur, and the existence of sensitive periods in development for such toxicity, has been undetermined. In terms of risk to the fetus, a potentially susceptible sub-population, there is concern, first, that the level of exposure at which neurotoxicity occurs may be different from the adult, and second, that the site of toxic action and subsequent neurotoxic effects of CO may be different in the immature and mature brain. The investigator studied the susceptibility of the developing brain to moderate levels of CO maintained chronically through the period of neuronal proliferation, and into the period of synapse formation. Carbon monoxide may be thought of as both a prototypical hypoxic agent, and a significant public health hazard in its own right. Carbon monoxide is a ubiquitous toxic agent that accounts for large numbers of deaths and significant morbidity in human populations. Subtle neurotoxic effects of this agent may be even more common, but they may go largely undetected, or fail to be associated with CO exposure. We have shown that prenatal CO exposure at moderate levels can produce significant neurotoxic effects in rats. The data obtained from the cerebellum and neostriatum, in particular, suggest that chronic, moderate perinatal CO exposure may disrupt neuronal proliferation and, perhaps, may disrupt certain markers for neurochemical transmission.

Abstract  The purpose of this study of 3211 cellulose-fiber production workers was to evaluate earlier findings of excess biliary tract and liver cancer in a similarly exposed cohort reported in 1990. Mortality from biliary tract and liver cancer was not increased in this study population, and there was no excess mortality from pancreatic cancer. Mortality was not elevated for cancers of the lung or liver, sites at which tumors were induced in experimental animals exposed to methylene chloride. Men with 20 or more years of employment exhibited increased mortality from prostate cancer, whereas women who also had 20 or more years of employment experienced higher-than-expected mortality from cervical cancer. Although these apparent increases in mortality are difficult to interpret biologically and are not consistent with previous studies, they require further investigation.

Abstract  DNA single-strand (ss) breaks were detected in the livers of B6C3F1 mice immediately following exposure to 4000-8000 p.p.m. methylene chloride (MC) for 6 h. This damage was undetectable 2 h after exposure, suggesting an active DNA repair process. Similarly, DNA ss breaks were detected in whole lung homogenates taken from mice exposed to 2000-6000 p.p.m. MC. The DNA of mouse Clara cells incubated in vitro with MC was also damaged at concentrations of 5 mM MC and above. Pre-treatment of mice with the glutathione depletor buthionine sulphoximine (BSO) caused a decrease in the amount of DNA damage detected, suggesting a GST-mediated mechanism. DNA damage was also reduced in Clara cells when incubated in vitro with MC in the presence of BSO. In CHO cells induction of DNA damage was dependent upon exogenous MC metabolism by mouse liver S100 fraction (but not microsomes) in the presence of GSH. DNA ss breaks were not induced by MC in hamster hepatocytes in vitro at concentrations from 5 to 90 mM MC, nor in eight individual samples of normal human hepatocytes exposed to MC at similar concentrations. The ability of MC to induce DNA ss breaks in the four species studied is entirely compatible with the known carcinogenicity of this chemical in animals and offers experimental evidence to suggest that humans would not be susceptible to MC-induced liver cancer. The DNA ss breaks correlate with the metabolism of MC by the GST pathway and provide an explanation for the lack of sensitivity of hamsters and rats to MC-induced liver cancer.

Abstract  Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.

Abstract  Mortality ascertainment was extended through 1990 for a cohort of 1271 workers involved in the production of cellulose triacetate fiber at a plant in Rock Hill, South Carolina. Each subject was employed for at least three months between 1 January 1954 and 1 January 1977 in jobs that entailed exposure to the highest concentrations of methylene chloride. Median exposures in 1977 ranged from 140 to 745 ppm (8-h time-weighted average). The observed numbers of deaths from specific causes were compared with the expected numbers of deaths computed from rates in York County, South Carolina. For most causes of death, there was little if any association with employment. Among causes of particular interest, no new deaths were observed from cancer of the liver and biliary tract, although the excess from the earlier study persisted (4 observed, 1.34 expected). No excess mortality was observed for cancer of the pancreas (2 observed, 2.42 expected) or for ischemic heart disease (43 observed, 47.8 expected).

Abstract  The objective of this study was to compare the human immunodeficiency virus (HIV) viral load (VL) and CD4 counts in patients infected with HIV with and without cervical cancer. The authors hypothesized that HIV-positive women with cervical cancer would have a greater risk of immune suppression.

A case-control study was conducted that included all HIV-positive patients who were seen at the authors' institution from January 1, 1995 to April 20, 2006 with invasive cervical cancer (cases) and without invasive cervical cancer (controls). Patients were included only if they had a CD4 count recorded<6 months before or<3 months after their diagnosis of invasive cervical cancer (cases) or at their last gynecologic examination (controls). Controls were matched to cases on a 4:1 ratio according to current smoking history. Patients were considered immunocompetent if they had both a CD4 count>200 cells/microL and a VL<10,000 copies/mL.

In total, 15 cases and 60 controls were identified. The median CD4 count for cases was 208 cells/microL (range, 18-1102 cells/microL) compared with 445 cells/microL (range, 20-1201 cells/microL) for controls (P=.03). The median VL was 16,918 copies/mL (range, 50-214,915 copies/mL) for cases compared with 1430 copies/mL (range, 50-571,000 copies/mL) for controls (P=.15). Only 1 of 14 cases (7%) was immunocompetent compared with 35 of 55 controls (64%; odds ratio, 0.04; 95% confidence interval, 0-0.37; P<.001). This significance was maintained after adjusting for other factors (P=.002).

Women with HIV who were diagnosed with invasive cervical cancer appeared to have a much greater degree of immunosuppression than women with HIV without invasive cervical cancer.

Abstract  More than a million workers are at risk for methylene chloride exposure. Aerosol sprays and paint stripping may also cause significant nonoccupational exposures. After methylene chloride inhalation, significant amounts of carbon monoxide are formed in vivo as a metabolic by-product. Poisoning predominantly affects the central nervous system and results from both carboxyhemoglobin formation and direct solvent-related narcosis. In this report, we describe a case of methylene chloride intoxication probably complicated by exogenous carbon monoxide exposure. The worker's presentation of intermittent headaches was consistent with both methylene chloride intoxication and carbon monoxide poisoning. The exposures and symptoms were corroborated by elevated carboxyhemoglobin saturations and a workplace inspection that documented significant exposures to both methylene chloride and carbon monoxide. When both carbon monoxide and methylene chloride are inhaled, additional carboxyhemoglobin formation is expected. Preventive efforts should include education, air monitoring, and periodic carboxyhemoglobin determinations. Methylene chloride should never be used in enclosed or poorly ventilated areas because of the well-documented dangers of loss of consciousness and death.

Abstract  The literature contains only one report of methylene chloride poisoning after ingestion. Unfortunately carboxyhaemoglobin (COHb) levels were not obtained in this case. We now describe a fatal case where a similar amount of the solvent was ingested and after which toxicology screening was done.

Abstract  A retrospective cohort mortality study was conducted among men employed for one or more years, between 1940 and 1969, at an operating division of a large chemical company. Vital status follow-up for the cohort of 1,919 men was determined through 1979 and identified 390 deaths. Overall mortality in the study group and in each of eight employment subgroups was less than that of the corresponding United States white male population. Additionally, standardized mortality ratios were not significantly elevated for any of the examined cause-of-death categories. Cause-specific mortality comparisons were also made among the employment subgroups and by duration of employment in the company division using an internal analysis method. There were no relationships observed for employment duration. Several significant differences (p less than 0.05) by employment subgroup were noted; however, neither the decreases nor increases presently could be ascribed to identifiable environmental factors.