Mark Toraason

DNA strand breaks, oxidative damage, and 1-OH pyrene in roofers with coal-tar pitch dust and/or asphalt fume exposure

OBJECTIVE To determine the potential for asphalt fume exposure to increase DNA damage, we conducted a cross-sectional study of roofers involved in the application of roofing asphalt. METHODS DNA strand breaks and the ratio of 8-hydroxydeoxyguanosine (8-OHdG) to 2-deoxyguanosine (dG) were measured in peripheral blood leukocytes of roofers. In addition, urinary excretion of 8-OHdG and 8-epi-prostaglandin F2alpha (8-epi-PGF) was also measured. The study population consisted of 26 roofers exposed to roofing asphalt and 15 construction workers not exposed to asphalt during the past 5 years. A subset of asphalt roofers (n = 19) was exposed to coal-tar pitch dust (coal tar) during removal of existing roofs prior to applying hot asphalt. Personal air monitoring was performed for one work-week to measure exposure to total particulates, benzene-soluble fraction of total particulates, and polycyclic aromatic compounds (PACs). Urinary 1-OH-pyrene levels were measured as an internal biomarker of PAC exposure. RESULTS Full-shift breathing zone measurements for total particulates, benzene-solubles and PACs were significantly higher for coal-tar exposed workers than for roofers not exposed to coal tar. Similarly, urinary 1-OH-pyrene levels were higher in coal-tar exposed roofers than roofers not exposed to coal tar. Total particulates or benzene-soluble fractions were not associated with urinary 1-OH-pyrene, but PAC exposure was highly correlated with urinary 1-OH-pyrene. When stratified by 1-OH-pyrene excretion, DNA strand breaks increased in a dose-dependent manner, and leukocyte 8-OHdG/dG decreased in a dose-dependent manner. Significant changes in DNA damage appeared to be linked to PACs from coal-tar exposure, although asphalt fume alone was associated with a small but significant increase in urinary 1-OH-pyrene and DNA strand breaks. CONCLUSIONS Results are consistent with previous reports that asphalt or coal-tar exposure can cause DNA damage. Urinary 8-epi-PGF remained relatively constant during the week for virtually all subjects, regardless of exposure indicating that neither asphalt nor coal-tar exposure induces an overt oxidative stress. A small, but statistically significant increase in 8OHdG was evident in end-of-week urine samples compared with start-of-week urine samples in roofers exposed to coal-tar. The increase in urinary 8OHdG coupled with the decrease in leukocyte 8-OHdG/dG, suggests that coal-tar exposure induces protective or repair mechanisms that result in reduced levels of steady-state oxidative-DNA damage.

Oxidative stress and DNA damage in Fischer rats following acute exposure to trichloroethylene or perchloroethylene.

Oxidative DNA damage is emerging as an biomarker of effect in studies assessing the health risks of occupational chemicals. Trichloroethylene (TCE) and perchloroethylene (PERC) are used in the dry cleaning industry and their metabolism can produce reactive oxygen compounds. The present study examined the potential for TCE and PERC to induce oxidative DNA damage in rats that was detectable as increased urinary excretion of 8-hydroxydeoxyguanosine (8OHdG). Thiobarbaturic acid reactive substances (TBARS) and 8-epiprostaglandin F2alpha (8epiPGF) were also measured as biomarkers of increased oxidative stress. Male Fischer rats were administered a single i.p. injection of 0, 100, 500, or 1000 mg/kg of PERC or TCE. Control rats received only vehicle (1:4 v/v of Alkamuls/water). A positive control group received 100 mg/kg 2-nitropropane (2NP). Rats were sacrificed 24 h after dosing. In rats receiving 2NP or TCE but not PERC, TBARS and the 8OHdG/dG ratios were significantly elevated in liver. Lymphocyte 8OHdG/dG was not affected significantly by 2NP, TCE or PERC. In rats receiving 2NP, urinary excretion of 8OHdG and 8epiPGF2 were significantly increased. In rats receiving TCE or PERC, significant increases in 8epiPGF2 or 8OHdG were not evident. Results indicate that a single high dose of TCE, but not PERC, can induce an increase in oxidative DNA damage in rat liver. However, the usefulness of 8OHdG as a biomarker of TCE-induced oxidative DNA damage is questionable.

Arachidonic acid disrupts calcium dynamics in neonatal rat cardiac myocytes

OBJECTIVES The purpose of this study was to investigate the effects of prolonged arachidonic acid (AA) exposure on electrically induced fluctuations of cytosolic free Ca2+ concentration ([Ca2+]i) in cardiac myocytes and to identify intracellular biochemical events that may play a role in the actions of AA on [Ca2+]i dynamics. METHODS Electrically induced [Ca2+]i transients were investigated in cultured single neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2-[Ca2+]i binding. KCl-induced depolarization, caffeine and ryanodine were used to assess the effects of AA on Ca2+ handling by the sarcolemma and the sarcoplasmic reticulum. Prostanoid formation was measured with an ELISA technique. alpha-Tocopherol was used to determine if free radical formation was a factor in the AA effects on [Ca2+]i. RESULTS Exposure to 10-30 microM AA produced a concentration-dependent and reversible configuration change and eventually a cessation of [Ca2+]i transients. Continued exposure resulted in a Ca2+ overload (tonic [Ca2+]i greater than peak systolic [Ca2+]i). AA did not influence KCl-induced [Ca2+]i increase but did eliminate caffeine-induced [Ca2+]i transients. AA exposure stimulated the formation of 6-oxo-prostaglandin F1 alpha in a concentration-dependent manner, but thromboxane B2 formation was not influenced. alpha-Tocopherol pretreatment significantly delayed times till cessation of [Ca2+]i transients and Ca2+ overload, whereas ryanodine and cyclo-oxygenase inhibitors were without effect. CONCLUSIONS The present data provide evidence that the initial action of AA on [Ca2+]i transients during excitation-contraction coupling involves an effect of AA on sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ handling. AA-induced cessation of electrically induced [Ca2+]i transients and Ca2+ overload may involve the formation of free radicals.

Effect of perchloroethylene, smoking, and race on oxidative DNA damage in female dry cleaners.

Perchloroethylene (PERC) is used widely as an industrial dry cleaning solvent and metal degreaser. PERC is an animal carcinogen that produces increased incidence of renal adenomas, adenocarcinomas, mononuclear cell leukemia, and hepatocellular tumors. Oxidative DNA damage and lipid peroxidation were assessed in 38 women with (dry cleaners) or without (launderers) occupational exposure to PERC. PERC exposure was assessed by collecting breathing zone samples on two consecutive days of a typical work week. PERC levels were measured in blood drawn on the morning of the second day of breathing zone sample collection in dry cleaners and before a typical workday in launderers. Blood PERC levels were two orders of magnitude higher in dry cleaners compared to launderers. A significant correlation was noted between time weighted average (TWA) PERC and blood PERC in dry cleaners (r=0.7355, P<0.002). 8-Hydroxydeoxyguanosine (8-OHdG), ng/mg deoxyguanosine (dG) in leukocyte nuclear DNA was used as an index of steady-state oxidative DNA damage. Urinary 8-OHdG, microg/g creatinine was used as an index of oxidative DNA damage repair. Urinary 8-epi-prostaglandin F(2alpha) (8-epi-PGF), ng/g creatinine was used as an index of lipid peroxidation. The mean+/-S.D. leukocyte 8-OHdG in launderers was 16.0+/-7.3 and was significantly greater than the 8.1+/-3.6 value for dry cleaners. Urinary 8-OHdG and 8-epi-PGF were not significantly different between dry cleaners and launderers. Unadjusted Pearson correlation analysis of log transformed PERC exposure indices and biomarkers of oxidative stress indicated a significant association in launderers between blood PERC and day 1 urinary 8-OHdG (r=0.4661, P<0.044). No significant associations between exposure indices and biomarkers were evident in linear models adjusted for age, body mass index, race, smoking (urinary cotinine, mg/g creatinine) and blood levels of the antioxidants Vitamin E and beta-carotene. The mean+/-S.D. leukocyte 8-OHdG value in control white women was 17.8+/-7.4 and was significantly greater than the 11.8+/-5.9 in control black women. No significant differences by race were evident for the other biomarkers. Smoking status was not significantly associated with any of the oxidative damage indices. Results indicate a reduction in oxidative DNA damage in PERC exposed dry cleaners relative to launderers, but PERC could not clearly be defined as the source of the effect.

Inhibition of rat heart mitochondrial electron transport in vitro: implications for the cardiotoxic action of allylamine or its primary metabolite, acrolein

Allylamine (3-aminopropene) is a specific cardiac toxicant that causes aortic, valvular and myocardial lesions in many species. Myocardial necrosis can be observed 24 h after a single dose. Acute toxicity is believed to involve metabolism of allylamine to highly reactive acrolein (2-propenal). Allylamine has been shown to bind to mitochondria from aorta and heart, suggesting that the subcellular site of injury is at or near the mitochondrion. The present investigation compared the effect of allylamine and its primary metabolite, acrolein, on electron transport and oxidative phosphorylation in mitochondria isolated from rat heart (RHM). Both compounds weakly inhibited mitochondrial electron transport with either the combination of glutamate, malate, and malonate (GMM, NADH-linked) or succinate as substrate. Comparisons of the slopes of concentration-effect regression (range of concentrations tested, 0.20-2.0 mM) lines showed acrolein to have significantly greater inhibitory effects than allylamine (range of concentrations tested, 0.22-6.4 mM) on GMM oxidation, while no significant difference in the abilities of the compounds to inhibit succinate oxidation were observed, indicating site preferences for inhibitory action. The addition of an uncoupling agent could not reverse inhibition with either substrate system. These results indicate that both the parent compound and its proposed metabolite primarily inhibit electron transport with little direct effect on the coupling mechanism. The State III EC50 (effective concentrations for 50% inhibition of control mitochondrial enzyme activities) for allylamine (2.29 mM with succinate as substrate and 1.22 mM with GMM) and acrolein (0.80 mM with succinate as substrate and 0.39 mM with GMM) are probably too great to invoke the direct action of either the parent compound or its oxidized metabolite on mitochondrial electron transport as a primary mechanism in the cardiotoxic action of allylamine.

Abrasive Blasting Agents: Designing Studies to Evaluate Relative Risk

Workers exposed to respirable crystalline silica used in abrasive blasting are at increased risk of developing a debilitating and often fatal fibrotic lung disease called silicosis. The National Institute for Occupational Safety and Health (NIOSH) recommends that silica sand be prohibited as abrasive blasting material and that less hazardous materials be used in blasting operations. However, data are needed on the relative risks associated with exposure to abrasive blasting materials other than silica. NIOSH has completed acute studies in rats (Hubbs et al., 2001; Porter et al., 2002). To provide dose-response data applicable to making recommendation for occupational exposure limits, NIOSH has collaborated with the National Toxicology Program (NTP) to design longer term studies with silica substitutes. For risk assessment purposes, selected doses will include concentrations that are relevant to human exposures. Rat lung burdens achieved should be comparable to those estimated in humans with working lifetime exposures, even if this results in “overloading” doses in rats. To quantify both dose and response, retained particle burdens in the lungs and lung-associated lymph nodes will be measured, as well as biochemical and pathological indices of pulmonary response. This design will facilitate assessment of the pulmonary fibrogenic potential of inhaled abrasive blasting agents at occupationally relevant concentrations.

Antimony-induced alterations in thiol homeostasis and adenine nucleotide status in cultured cardiac myocytes

Cultured cardiac myocytes were exposed for up to 4 h to 50 and 100 microM potassium antimonyl tartrate (PAT). After 4 h, 50 and 100 microM PAT killed 14 and 33% respectively of the cardiac myocytes. PAT-induced alterations in both protein and nonprotein thiol homeostasis. Transient increases in oxidized glutathione disulfide (GSSG) levels were detected after cells were treated with 100 microM PAT for 2 h. After 4 h, both concentrations of PAT significantly depleted reduced glutathione (GSH) levels. Protein thiols levels were also decreased after a 2-h exposure to 50 and 100 microM PAT. Cells treated with 50 microM and 100 microM PAT had a 15% and 40% reduction respectively in protein thiols after 4 h. PAT also significantly inhibited glutathione peroxidase and pyruvate dehydrogenase activity in cardiac myocytes. Pyruvate dehydrogenase activity levels were inhibited as early as 1 h after cells were treated with both concentrations of PAT. Cardiac myocyte ATP levels were also decreased by PAT, but only after a 4-h exposure to 50 microM and 100 microM PAT. Decreases in cellular ATP levels paralleled PAT toxicity put appeared to be secondary to other cellular changes initiated by PAT exposure.

Ethylene Glycol Monomethyl Ether (EGME) Inhibits Rat Embryo Ornithinf Decarboxylase (ODC) Activity

The effects of ethylene glycol monomethyl ether (EGME) on reproductive outcome in the rat, and on ornithine decarboxylase (ODC) activity in the rat embryo were evaluated. Dams (n = 8) were treated by gavage on gestation days 6-12 (sperm = day 0) with 0, 25, 50 or 75 mg/kg EGME in 10 ml/kg distilled water. EGME had a dose-dependent effect on reproductive outcome. Gestation length was prolonged, and the number of litters delivered and neonatal body weight were reduced. Whole embryo ODC was measured on gestation days 9, 11, 13 and 15. ODC attained maximum activity in controls on day 11, increasing by more than an order of magnitude above the activity found on day 9. On day 11, a statistically significant dose-dependent inhibition of ODC activity was observed with the maximum dose of EGME inhibiting ODC activity 60 percent. On days 13 and 15, ODC activity declined markedly from peak values, and the dose-dependent inhibition was no longer evident. The study demonstrates a correlation between the inhibition of embryonic ODC activity by EGME and the effect of EGME on reproductive outcome.

Prenatal ethylene glycol monomethyl ether (EGME) exposure produces electrocardiographic changes in the rat

The purpose of the present study was to determine if electrocardiographic (EKG) changes observed in fetuses exposed in utero to ethylene glycol monomethyl ether (EGME) persisted beyond the fetal/neonatal period. Groups of pregnant Sprague-Dawley rats were gavaged on gestation days 7-13 (sperm = day 0) with 0, 50, or 75 mg/kg EGME. Body weight prior to delivery was reduced and gestation was prolonged in EGME-treated dams. EGME treatment reduced the percentage of pregnant dams that delivered, litter size, and pup weight. There were no survivors beyond 3 days of age in the 75 mg/kg EGME group. The number of litters surviving through weaning and weight gain of male and female offspring through 8 weeks of age were reduced in the 50 mg/kg EGME group. In this same group, heart weight was unaffected, but heart/body weight ratios were increased when rats were 8 weeks old. EKGs were obtained from unanesthetized and unrestrained rats at 3 and 6 weeks of age. Prenatal EGME exposure increased the QRS interval in 3- and 6-week-old rats, and increased the T wave in 6-week-old rats. Thirty-six and 54% of 3- and 6-week old litters, respectively, had one or more individuals that were classified as having an intraventricular conduction delay (double R wave and QRS interval of 14 msec or longer). No microscopic heart abnormalities were associated with the observed intraventricular conduction delay.

Depression of contractility in cultured cardiac myocytes from neonatal rat by carbon tetrachloride and 1,1,1-trichloroethane☆

The cardiac depressant effects of carbon tetrachloride (CCl(4)) and 1,1,1-trichloroethane (CH(3)CCl(3)) were evaluated in cultured heart cells from neonatal rats. Heart cells were grown on glass coverslips and formed a confluent monolayer that beat spontaneously, rhythmically and in synchrony. Contractility was assessed by video-motion analysis. Stock solutions of CCl(4) or CH(3)CCl(3) were prepared in dimethylsulphoxide (DMSO) and aliquoted (final DMSO concentration 0.2%) into medium (M199 supplemented with 5% serum) immediately prior to perfusion across myocytes in an environmentally controlled chamber. CCl(4) and CH(3)CCl(3) had a negative chronotropic effect on myocytes by prolonging the relaxation phase of beating. Duration of the contraction phase of beating, and peak velocity of cell wall movement were not affected by these halocarbons. Beating was stopped by 2.5 mm-CCl(4) or 5 mm-CH(3)CCl(3), and washout of these compounds resulted in a resumption of beating activity. Increasing (3.6 mm) or decreasing (0.6 mm) the calcium concentration of the medium (normal = 1.8 mm) significantly affected the duration of contraction and relaxation phases of beating, but did not alter the concentration-dependent action of CCl(4). A positive chronotropic effect of isoproterenol was evident from 10(-9) to 10(-6)m, but contractility was depressed by isoproterenol concentrations greater than 10(-8)m in the presence of 750 mum-CCl(4). This study demonstrates the usefulness of cultured heart cells for assessing the cardiac depressant and sensitizing actions of halogenated hydrocarbons.