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1-Bromopropane (1-BP; n -propyl bromide) (CAS No. 106-94-5) is an alternative to ozone-depleting chlorofluorocarbons that has a variety of potential applications as a degreasing agent for metals and electronics, and as a solvent vehicle for spray adhesives. Its isomer, 2-brompropane (2-BP; isopropyl bromide) (CAS No. 75-26-3) impairs antioxidant cellular defenses, enhances lipid peroxidation, and causes DNA damage in vitro. The present study had two aims. The first was to assess DNA damage in human leukocytes exposed in vitro to 1- or 2-BP. DNA damage was also assessed in peripheral leukocytes from workers with occupational exposure to 1-BP. In the latter assessment, start-of- and end-of-work week blood and urine samples were collected from 41 and 22 workers at two facilities where 1-BP was used as a solvent for spray adhesives in foam cushion fabrication. Exposure to 1-BP was assessed from personal-breathing zone samples collected for 1–3 days up to 8 h per day for calculation of 8 h time weighted average (TWA) 1-BP concentrations. Bromide (Br) was measured in blood and urine as a biomarker of exposure. Overall, 1-BP TWA concentrations ranged from 0.2 to 271 parts per million (ppm) at facility A, and from 4 to 27 ppm at facility B. The highest exposures were to workers classified as sprayers. 1-BP TWA concentrations were statistically significantly correlated with blood and urine Br concentrations. The comet assay was used to estimate DNA damage. In vitro, 1- or 2-BP induced a statistically significant increase in DNA damage at 1 mM. In 1-BP exposed workers, start-of- and end-of-workweek comet endpoints were stratified based on job classification. There were no significant differences in DNA damage in leukocytes between workers classified as sprayers (high 1-BP exposure) and those classified as non-sprayers (low 1-BP exposure). At the facility with the high exposures, comparison of end-of-week values with start-of-week values using paired analysis revealed non-sprayers had significantly increased comet tail moments, and sprayers had significantly increased comet tail moment dispersion coefficients. A multivariate analysis included combining the data sets from both facilities, log transformation of 1-BP exposure indices, and the use of multiple linear regression models for each combination of DNA damage and exposure indices including exposure quartiles. The covariates were gender, age, smoking status, facility, and glutathione S -transferase M1 and T1 (GSTM1, GSTT1) polymorphisms. In the regression models, start-of-week comet tail moment in leukocytes was significantly associated with serum Br quartiles. End-of-week comet tail moment was significantly associated with 1-BP TWA quartiles, and serum Br quartiles. Gender, facility, and GSTM1 had a significant effect in one or more models. Additional associations were not identified from assessment of dispersion coefficients. In vitro and in vivo results provide limited evidence that 1-BP exposure may pose a small risk for increasing DNA damage.

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.

A workshop convened to define research needs in toxicology identified several deficiencies in data and methods currently applied in risk assessment. The workshop panel noted that improving the link between chemical exposure and toxicological response requires a better understanding of the biological basis for inter-and intra-human variability and susceptibility. This understanding will not be complete unless all life stages are taken into consideration. Because animal studies serve as a foundation for toxicological assessment, proper accounting for cross-species extrapolation is essential. To achieve this, adjustments for dose-rate effects must be improved, which will aid in extrapolating toxicological responses to low doses and from short-term exposures. Success depends on greater use of validated biologically based dose-response models that include pharmacokinetic and pharmacodynamic data. Research in these areas will help define uncertainty factors and reduce reliance on underlying default assumptions....

While less than 1% of the general population is sensitized to latex, the U.S. Occupational Safety and Health Administration estimates that 8-12% of health-care workers are sensitized. The major source of workplace exposure is powdered natural rubber latex (NRL) gloves. NRL is harvested from HEVEA: brasiliensis trees and ammoniated to prevent coagulation resulting in the hydrolysis of the latex proteins. Prior to use in manufacturing, the latex is formulated by the addition of multiple chemicals. Thus, human exposure is to a mixture of residual chemicals and hydrolyzed latex peptides. Clinical manifestations include irritant contact dermatitis, allergic contact dermatitis (type IV), and type I immediate hypersensitivity response. Type I (IgE-mediated) NRL allergy includes contact urticaria, systemic urticaria, angioedema, rhinitis, conjunctivitis, bronchospasm, and anaphylaxis. Taking an accurate history, including questions on atopic status, food allergy, and possible reactions to latex devices makes diagnosis of type-I latex allergy possible. To confirm a diagnosis, either in vivo skin prick testing (SPT) or in vitro assays for latex-specific IgE are performed. While the SPT is regarded as a primary confirmatory test for IgE-mediated disease, the absence of a U.S. Food and Drug Administration-licensed HEVEA: brasiliensis latex extract has restricted its use in diagnosis. Serological tests have, therefore, become critically important as alternative diagnostic tests. Three manufacturers currently have FDA clearance for in vitro tests, to detect NRL-specific IgE. The commercially available assays may disagree on the antibody status of an individual serum, which may be due to the assay's detecting anti-NRL IgEs to different allergenic NRL proteins. Sensitized individuals produce specific IgE antibody to at least 10 potent HEVEA: allergens, Hev b 1-Hev b 10, each of which differs in its structure, size, and net charge. The relative content and ratios of Hevs in the final allergen preparation most probably could effect diagnostic accuracy. The Hev proteins have been cloned and expressed as recombinant proteins. Sequencing demonstrates both unique epitopes and sequences commonly found in other plant proteins. Sequence homology helps to explain the cross reactivity to a variety of foods experienced by latex allergic individuals. The development of recombinant allergens provides reagents that should improve the diagnostic accuracy of tests for latex allergy. Although clinical and exposure data have been gathered on the factors affecting response in latex-allergic individuals, less is known regarding the development of sensitization. Coupled with in vitro dermal penetration studies, murine models have been established to investigate the route of exposure in the development of latex sensitization. Time-course and dose-response studies have shown subcutaneous, intratracheal, or topical administrations of non-ammoniated latex proteins to induce IgE production. Both in vitro penetration and in vivo studies highlight the importance of skin condition in the development of latex allergy, with enhanced penetration and earlier onset of IgE production seen with experimentally abraded skin. The diagnosis of latex allergy is complicated by these variables, which in turn hinder the development of intervention strategies. Further epidemiological assessment is needed to more explicitly define the scope, trends, and demographics of latex allergy. Diagnostic accuracy can be improved through greater knowledge of proteins involved in the development of latex allergy, and better documentation of the presently available diagnostic tests. In vivo and in vitro models can elucidate mechanisms of sensitization and provide an understanding of the role of the exposure route in latex allergy-associated diseases. Together, these efforts can lead to intervention strategies for reducing latex allergy in the workplace.

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.

Cardiac myocytes were exposed to concentrations of potassium antimonyl tartrate (PAT) ranging from 1 to 1000 microM for 1 to 24 hr. Toxicity was assessed by measuring lactate dehydrogenase (LDH) release and by monitoring chronotropic depression. Lipid peroxidation was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). PAT produced a concentration- and time-dependent depression in chronotropy and an increase in the release of LDH and TBARS. A 4-hr exposure to 100 microM PAT stopped beating and induced significant increases in TBARS and LDH release in the myocyte cultures. The lipid peroxidation and LDH release induced by 100-200 microM PAT at 4 hr could be prevented by pretreatment of the cardiac myocytes with vitamin E or by the simultaneous addition of other antioxidants. Vitamin E continued to protect against lipid peroxidation up to 18 hr after the addition of 100 microM PAT, but failed to provide significant protection against LDH release at this time-point. Both 50 and 100 microM PAT decreased cardiac myocyte glutathione (GSH) levels after a 4-hr exposure. A series of thiol-containing compounds was evaluated for their effects on PAT toxicity. The addition of dithiothreitol, GSH, and 2-mercaptoethanol afforded some degree of protection against lipid peroxidation and LDH release up to 18 hr after the addition of 100 microM PAT. These results suggest that PAT induces lipid peroxidation in cultured cardiac myocytes but that other mechanisms may contribute to cell death with long-term exposures to PAT. Our results also suggest that PAT interacts with thiol-containing compounds.

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 m m -CCl 4 or 5 m m -CH 3 CCl 3 , and washout of these compounds resulted in a resumption of beating activity. Increasing (3.6 m m ) or decreasing (0.6 m m ) the calcium concentration of the medium (normal = 1.8 m m ) 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 μ m -CCl 4 . This study demonstrates the usefulness of cultured heart cells for assessing the cardiac depressant and sensitizing actions of halogenated hydrocarbons.

The purpose of the present study was to examine whether cardiac actions of dichloromethane (DCM) in vivo correlate with in vitro alterations of Ca(2+) dynamics in cardiac myocytes. Electrically induced fluctuations of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) were investigated in neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2 binding. [Ca(2+)](i) transients were inhibited in a concentration-dependent and reversible manner with IC(10) and IC(50)values of 3.2 and 18.1 mm. Complete inhibition of [Ca(2+)](i) transients and cessation of beating were observed at 40.96 mm without morphological alterations. Left ventricular pressure in urethane-anaesthetized rats was measured by introducing a tip catheter by way of the carotid artery into the left ventricle and ECG (lead II) was recorded by two needle electrodes. Administration of 3.1, 6.2 or 12.4 mmol DCM/kg orally resulted in DCM blood concentrations between 1.0 and 1.6 mm accompanied by a dose-dependent decrease of contractility parameters. Moreover, DCM administration provided protection against arrhythmia development due to CaCl(2) infusion. These observations are consistent with the view that both the negative inotropic effects of DCM and the protection from CaCl(2)-induced arrhythmia are mediated by an inhibition of Ca(2+) dynamics in cardiomyocytes.

Inhibition of intercellular communication is proposed to be one of several possible mechanisms of teratogenesis. 38 coded compounds were tested for their effect on intercellular communication in the V79 cell metabolic co-operation assay. Test chemicals were selected from a list of 47 agents recommended for the evaluation of assays in vitro for developmental toxicants. In addition to testing the effects of chemicals on intercellular communication, a separate cytotoxicity assay determined the concentration of each chemical that inhibited clonal expansion of V79 cells. Seven of the 29 designated teratogens were positive for inhibition of intercellular communication in the V79 assay. Additionally, four teratogens and one non-teratogen inhibited intercellular communication at only a single concentration or at cytotoxic concentrations and were scored as equivocal. Therefore, the sensitivity of the V79 assay for teratogens was 24% (seven of 29 teratogens tested positive), or 38% if the four equivocal chemicals are considered positive. None of the nine non-teratogens unequivocally inhibited intercellular communication, resulting in a specificity of 100%, which decreased to 89% when the single equivocal score was considered positive. The overall accuracy for correctly identifying teratogens and non-teratogens was 42% when equivocal chemicals were considered negative, and 50% if they were considered positive in the V79 assay. The results demonstrate that despite relatively low accuracy regarding a diverse group of developmental toxicants, chemicals that did inhibit intercellular communication under the present conditions had a high probability of being a teratogen. The low accuracy reported here contrasts with earlier reports on the assay and possible reasons for this are discussed.

We examined the effect of 11 aliphatic halogenated hydrocarbons on the transfer of microinjected dye among cardiac myocytes from neonatal rats. Myocytes were suffused with increasing concentrations of halocarbon added as a 0.2% solution of dimethyl sulfoxide to M199 containing 1.8 m m Ca and 5% serum. Single cells were microinjected with the fluorescent probe Lucifer yellow (5% in 0.1 m m LiCl) and dye coupling to adjacent cells was monitored. All of the halocarbons tested exhibited a concentration-dependent inhibitory effect on intercellular communication that was reversible following washout of the compounds. Intercellular communication was blocked within 1 min of exposure to an effective concentration, and recovery of communication occured even after 2 hr of exposure. Pretreatment of cells with SKF 525-A (25 μM) did not prevent the inhibition of intercellular communication by carbon tetrachloride suggesting an absence of P-450 involvement. EC50s were calculated for each chemical using probit analysis. A log-log comparison of the EC50s and the physicochemical properties of the chemicals demonstrated a high correlation ( R 2 = 0.933) between the EC50s and the octanol/water partition coefficients of the halocarbons. This suggests that incorporation of halocarbons in the membrane may block intercellular communication though modification of the immediate environment of the gap junctions. The results are in agreement with the hypothesis that inhibition of gap junctional communication is a factor in the arrhythmogenic effects of acute halocarbon exposure.

Asphalt fume condensate is a skin carcinogen in mice, yet this complex mixture contains relatively low levels of known carcinogenic initiators. Consequently, its biological activity has been attributed to the presence of cocarcinogenic or tumor-promoting agents. One of several proposed mechanisms of tumor promotion is inhibition of intercellular communication. In an attempt to determine if asphalt fume has tumor-promoting potential inhibition of intercellular communication was measured in V79 cells exposed to fractionated asphalt fume condensate. Fume from air-blown Arabian crude asphalt was trapped and separated into five fractions by preparative-scale high-pressure liquid chromatography. The parent fume condensate and the five fractions inhibited intercellular communication in a concentration-dependent fashion, with a minimum effective concentration of 2.5 microgram/ml for the most potent fraction. Cytotoxicity assays were performed at the same time and concentrations as the metabolic cooperation assays. Cytotoxic responses paralleled the inhibition of intercellular communication.

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, P0.002). 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, P0.044). 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.

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.

The effects of nonlethal concentrations of potassium antimonyl tartrate (PAT) were examined in cultured neonatal rat cardiac myocytes. PAT (5, 10 microM) significantly increased cellular reduced glutathione (GSH) and heme oxygenase activity after 18 h. GSH levels and heme oxygenase activity were increased 2.5- and 5.4-fold, respectively, by 10 microM PAT after 18 h. In addition, total cytochrome P450 levels were decreased by PAT after an 18-h exposure. PAT exposures were associated with the induction of specific stress proteins. Nonlethal concentrations of PAT produced a dose-dependent increase in HO-1, HSP70, and HSP25/27 protein levels but did not increase HSP60 levels. Pretreatment of cardiac myocytes with low concentrations of PAT (0.5-10 microM) protected against a subsequent lethal concentration of PAT (200 microM). This protection was blocked if cells were treated with the protein synthesis inhibitor cycloheximide. Results demonstrate that low concentrations of PAT increase GSH levels and stress protein synthesis, which may be responsible for the protection that low-level PAT exposure offers against the subsequent toxicity of higher concentrations of PAT.

Industrial exposure and pharmaceutical use of antimony compounds have been linked to altered cardiovascular function and pathology. Antimony compounds induce hypotension, bradycardia, and cardiac arrhythmias, all of which can arise from aberrations in myocyte regulation of intracellular free calcium concentration ([Ca 2+ ] i ). To determine if trivalent antimony affects [Ca 2+ ] i during excitation–contraction, we developed an in vitro cardiac myocyte model that was exposed for 24 hr to potassium antimonyl tartrate (PAT) at 0–10 μ m . Control myocytes received sodium potassium tartrate. Concentrations of up to 10 μ m PAT were without effect on total DNA and protein content of cultures, indicating that PAT exposures were not overtly toxic. However, spontaneous beating rates of myocytes were significantly reduced by 5 and 10 μ m PAT. Myocytes were paced by electric field stimulation at 0.5 Hz, and the effect of PAT on [Ca 2+ ] i transients during excitation–contraction was monitored with fura-2. PAT (2–8 μ m ) significantly reduced systolic [Ca 2+ ] i in a concentration-dependent fashion, but was without effect on diastolic [Ca 2+ ] i or on the first derivative of the transient rise ( d [Ca 2+ ] i / dt ). Myocytes from control cells responded to epinephrine (10 −8 –10 −5 m ) in concentration-dependent fashion with elevated systolic [Ca 2+ ] i and an increase in the rate of decay of transients. In PAT-exposed myocytes, the systolic response was blunted while the decay rate was enhanced. PAT-exposed cells also exhibited a reduced basal [Ca 2+ ] i when depolarized by 90 m m KCl and a reduced caffeine-releasable Ca 2+ pool of the sarcoplasmic reticulum. Both control and PAT-treated cells responded to ryanodine in a comparable fashion. Results indicate that a nonlethal exposure to PAT reduces Ca 2+ availability during excitation–contraction. Decreased influx of Ca 2+ across the sarcolemma and enhanced removal of Ca 2+ appear to be responsible.

Trivalent antimony, delivered as potassium antimonyl tartrate (PAT), has been previously shown to induce an oxidative stress and toxicity in cultured neonatal rat cardiac myocytes. The present study investigates the effect of PAT on intracellular free calcium ([Ca2+]i), which has been implicated in the toxicity of agents inducing oxidative stress, and explores its role in PAT toxicity. Exposure to 50 or 200 microM PAT led to progressive elevation in diastolic or resting [Ca2+]i and eventually a complete loss of [Ca2+]i transients that occurred well before cell death as assessed by LDH release. Prior loading of myocytes with the intracellular calcium chelator BAPTA (10 to 40 microM), protected against PAT toxicity in the presence and absence of extracellular calcium, and demonstrated a crucial role for [Ca2+]i in PAT toxicity. Exposure to 200 microM PAT in the absence of extracellular calcium slightly elevated [Ca2+]i, but only to levels comparable to resting [Ca2+]i for cells in 1.8 mM extracellular calcium. This demonstrated that although PAT toxicity was dependent on [Ca2+]i, a large increase above resting levels was not needed, and also that some calcium was mobilized from intracellular stores. However, the caffeine-releasable pool of sarcoplasmic reticulum calcium was increased, not depleted, by exposure to 200 microM PAT. These results demonstrate that PAT disrupts [Ca2+]i handling and support a role for a calcium-dependent event, but do not support the necessity of events in PAT-induced cell death that are mediated by a large elevation in [Ca2+]i.

The purpose of the present study was to determine if cardiac actions of dichloromethane (DCM) in vivo correlate with in vitro alterations of Ca2+ dynamics in cardiac myocytes. Neonatal rat ventricular myocytes were obtained from 2- to 4-day-old rats, and electrically induced fluctuations of cytosolic free Ca2+ concentration ([Ca2+]i) in single cardiomyocytes were investigated using spectrofluorometric analysis of fura-2-[Ca2+]i binding. In cultured myocytes, cumulative exposure to 0.64–40.96 mM DCM resulted in a concentration-dependent and reversible decrease in the magnitude of [Ca2+]i transients with IC10 and IC50 values of 7.98 and 18.82 mM, respectively. Total inhibition of [Ca2+]i transients and cessation of beating were observed at 40.96 mM DCM. Suffusion with DCM for 40 min did not cause morphological alterations of the myocytes. In a urethane-anesthetized rat model, left ventricular pressure was measured by introducing a tip catheter via the carotid artery into the left ventricle, the ECG was recorded by two needle electrodes applied subcutaneously to the chest wall, and arterial pressure was measured via the femoral artery. Oral administration of 3.1–12.4 mmol DCM/kg resulted in DCM blood concentrations between 1.0 and 1.6 mM, accompanied by a dose-dependent decrease in contractile force and heart rate without influencing blood pressure and ECG tracings. Moreover, DCM treatment provided significant protection against arrhythmia development due to CaCl2-infusion. In spite of the slight discrepancy between DCM blood concentrations and in vitro concentrations of DCM for [Ca2+]i transient inhibition, present data are consistent with the view that cardiac effects after DCM exposure are mediated by alterations of Ca2+ dynamics during excitation-contraction coupling.

Free radical-induced oxidative stress has been linked to ischemia-reperfusion injury of the myocardium. The .OH radical is considered the most damaging radical and can be increased in cells by treatment in vitro with H2O2. The purpose of the present study was to determine if aliphatic halocarbons enhance H2O2-induced oxidative injury in isolated cardiac myocytes from neonatal rats. Oxidative damage was assessed by measuring release of thiobarbituric acid-reactive substances (TBARS) from lipid peroxidation, loss of lactate dehydrogenase (LDH) through damaged sarcolemmal membranes, and alterations in intracellular calcium ([Ca2+]i) transients in electrically stimulated (1 Hz, 10 ms, 60 V) myocytes. H2O2 increased TBARS release and LDH leakage in a concentration-dependent (20-200 microM) manner. Continuous suffusion with H2O2 first altered the configuration of [Ca2+]i transients, then eliminated them, and finally caused [Ca2+]i overload (basal [Ca2+]i exceeded peak systolic [Ca2+]i of control). The time to [Ca2+]i overload was inversely associated with concentration, and the shortest time to overload was obtained with 100 microM H2O2. A 1-h preincubation of myocytes with the iron chelator deferoxamine inhibited all effects of H2O2. 1,1,1-Trichloroethane, carbon tetrachloride, or halothane at 1 mM significantly and reversibly reduced [Ca2+]i transients but did not influence TBARS release or LDH leakage. Simultaneous exposure of myocytes to H2O2 and halocarbons did not affect the myocyte response to H2O2 exposure. Results indicate that the three halocarbons tested do not enhance H2O2-induced oxidative injury in isolated cardiac myocytes.

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 EC 50 (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.

Allylamine is toxic to the cardiovascular system causing aortic, valvular and myocardial lesions. Acute toxicity is believed to involve metabolism of allylamine to highly reactive acrolein. Comparative toxicity of allylamine and acrolein was evaluated in cardiac fibroblasts and myocytes, which were obtained from neonatal rat hearts by a differential plating technique. Allylamine and acrolein were added directly to serum supplemented culture media (M199). Toxicity was assessed by measuring lactate dehydrogenase (LDH) release as an indicator of cell lysis. Spontaneous beating activity of myocytes, and adenosine 5' triphosphate (ATP) levels of myocytes and fibroblasts were also assessed. Cell lysis occurred 4 h after treatment of myocytes with 0.5 mM allylamine, whereas 20 mM allylamine was required to lyse fibroblasts. Acrolein, at a concentration of 0.05 mM, was equally toxic to fibroblasts and myocytes. Semicarbazide, a benzylamine oxidase inhibitor, protected myocytes from allylamine toxicity, but clorgyline, a monoamine oxidase inhibitor, was ineffective. Semicarbazide was ineffective against acrolein toxicity. Beating activity of myocytes was arrested by 0.05 mM acrolein and 0.5 mM allylamine, although 0.05-0.1 mM allylamine reduced beating activity. Myocyte ATP levels were reduced 4 h after exposure to 0.01 mM acrolein. Allylamine at 0.05 mM reduced ATP in myocytes, but 10 mM allylamine was required to reduce ATP in fibroblasts. ATP levels remained normal in myocytes exposed to 1 mM allylamine in the presence of 0.1 mM semicarbazide. The findings support the hypothesis that the toxicity of allylamine in cultured myocytes is dependent on its metabolism to acrolein, and that cytotoxicity may result from interference with energy production.

It has been suggested that calcium and cadmium compete for an intestinal transport system that is vitamin D dependent. To further test this hypothesis, the interaction between calcium and cadmium during transport in the duodenum of rat was investigated. Control rats maintained on a diet adequate in vitamin D, calcium, and phosphorus were compared to rats on the same diet administered 500 ng of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) 4 hr before transport measurement with everted sacs. Active transport of calcium was evident in control rats and was further stimulated by exogenous 1,25(OH)2D3. Added to mucosal bathing fluid 10 micron cadmium partially inhibited active calcium transport both in controls and in rats receiving 1,25(OH)2D3; 100 microM cadmium completely blocked active transport in both groups. Water transport was also inhibited by 10 and 100 microM cadmium. Cadmium uptake and transport were not affected by 1,25(OH)2D3. The accumulation of cadmium in mucosal tissue was significantly inhibited by 1 mM calcium, but there was no significant effect on uptake or transmural transport. The findings suggest that cadmium and calcium do not interact specifically at a 1,25(OH)2D3-dependent transport site. The interaction between calcium and cadmium in the duodenal mucosa could be related to the action of cadmium in blocking active transport processes.

The widely used industrial solvent ethylene glycol monomethyl ether (EGME) is teratogenic to rats and mice, inducing a variety of heart and major vessel abnormalities. In the present study, electrocardiography was used to evaluate heart function in day 20 rat (Sprague-Dawley) fetuses from mothers treated on gestation days 7-13 (sperm = day 1) with 0, 25, or 50 mg/kg EGME by gavage in 10 ml/kg water. The increased incidence of fetuses with cardiovascular malformations (primarily right ductus arteriosus and ventricular septal defect) and abnormal electrocardiograms (EKG) was dose dependent. The most prevalent EKG abnormality was a prolonged QRS wave. Mean QRS intervals were not significantly increased by EGME exposure, but there were significantly more litters in the 50-mg/kg EGME group that had one or more fetuses with QRS complexes of 40 msec or longer. The enhanced duration and the appearance of the aberrant QRS's suggested the presence of an intraventricular conduction delay in these fetuses. Heart rate and other EKG characteristics such as the P wave or P-R and Q-T intervals were not significantly affected by exposure to EGME. There did not appear to be an association between abnormal EKG's and fetal heart dysmorphology.

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.

In mammals, ornithine decarboxylase (ODC) activity is highest during periods of rapid cellular growth and development, and the normal pattern of ODC activity during this period is sensitive to chemical and drug exposure. The industrial solvent ethylene glycol monomethyl ether (EGME) is teratogenic to rats and mice, with the heart being particularly sensitive. Basal ODC activity and ODC activity following an isoproterenol challenge were used to assess heart function in 3-, 9-, 16- and 22-day-old offspring from dams treated with 25 mg/kg EGME by gavage on days 7-13 or 13-19 of gestation. Reproductive outcome was not affected by EGME and none of the offspring had gross physical abnormalities. Gestation length was prolonged by both EGME treatments, but the increase was statistically significant only in the group treated on days 7-13 gestation. ODC activity per mg protein was greatest in 3-day-old rats and dropped off sharply during the following 3 weeks. In 3-day-old rats exposed on days 7-13 of gestation, ODC activity was 54% of that found in controls. ODC activity was comparable to that in controls in 3-day-old rats exposed on days 13-19 of gestation. Isoproterenol increased ODC activity in all groups, but additional functional abnormalities were not revealed by the isoproterenol challenge.

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.

The industrial solvent ethylene glycol monomethyl ether (EGME) is a known teratogen that has been reported to alter calcium metabolism in guinea pigs during chronic exposure. Because of the tremendous demand of reproduction on maternal calcium stores, the effects of EGME on calcium and vitamin D metabolism during gestation were examined. Timed pregnant rats were treated by gavage with 0, 50, or 100 mg/kg EGME in 10 ml/kg distilled water on Days 9–15 of gestation (sperm = Day 1) and examined on Days 16 and 21. Virgin rats were treated for 7 days with 0 or 100 mg/kg EGME and examined 5 days later. EGME exposure did not affect body or kidney weight in virgin or pregnant rats, but liver weight was reduced in near-term pregnant rats treated with 100 mg/kg EGME. EGME (50 mg/kg) reduced litter size and fetal body weight and caused a significant number of live fetuses to have visceral abnormalities. EGME (100 mg/kg) caused all fetuses to be resorbed. In nonpregnant rats, 100 mg/kg did not affect serum 1,25-dihydroxyvitamin D (1,25(OH) 2 D 3 ) 25-hydroxyvitamin D, ionic calcium, total calcium, or parathyroid hormone. EGME appeared to have a dose-dependent effect on calcium and vitamin D metabolism during gestation. On Day 21 of gestation, total calcium and ionic calcium were increased and 1,25(OH) 2 D 3 was reduced in rats treated with EGME compared with nontreated controls. However, significant alterations in calcium homeostasis were evident only in dams that completely resorbed their litters. The changes in calcium and vitamin D metabolism during gestation appear to be secondary to the EGME-induced loss of litters.