Mitochondria have long been known to participate in the process of cell injury associated with metabolic failure. Only recently, however, have we come to appreciate the role of mitochondria as primary intracellular targets in the initiation of cell dysfunction. In addition to ATP synthesis, mitochondria are also critical to modulation of cell redox status, osmotic regulation, pH control, and cytosolic calcium homeostasis and cell signaling. Mitochondria are susceptible to damage by oxidants, electrophiles, and lipophilic cations and weak acids. Chemical-induced mitochondrial dysfunction may be manifested as diverse bioenergetic disorders and considerable effort is required to distinguish between mechanisms involving critical mitochondrial targets and those in which mitochondrial dysfunction is secondary and plays only a modulatory role in cell injury. The following paragraphs review a few important examples of chemical-induced cytotoxic responses that are manifested as interference with mitochondrial metabolism and bioenergetics, gene regulation, or signal transduction in the form of apoptosis and altered cell cycle control. Greater understanding of the molecular mechanisms of mitochondrial bioenergetics, ion regulation, and genetics will lead to numerous additional examples of mitochondria-mediated cell injury, revealing important new insight regarding the prediction, prevention, diagnosis, and treatment of chemical-induced toxic tissue injury.
Our laboratory has developed a method of intratracheal inhalation whereby rats can be exposed to high aerosol concentrations, resulting in high lung particle burdens in a short time period with deposition occurring directly in the lower respiratory tract, thus avoiding many drawbacks of larger nose-only or whole body inhalation systems. In this report, we compare the response of rats exposed by intratracheal inhalation to "fine" (approximately 250 nm) and "ultrafine" (approximately 21 nm) titanium dioxide particles with rats exposed to similar doses by intratracheal instillation. Animals receiving particles through inhalation showed a decreased pulmonary response, measured by bronchoalveolar lavage parameters, in both severity and persistence, when compared with those receiving particles through instillation. These results demonstrate a difference in pulmonary response to an inhaled vs an instilled dose, which may be due to differences in dose rate, particle distribution, or altered clearance between the two methods.
The database of Continuous Breeding mouse studies was evaluated to determine the relationships between the functional indicators of reproduction (pup measures) and the various necropsy endpoints collected for males and females. Of 72 chemicals studied, both males and females were affected in 33 studies, while females and/or conceptuses were affected in 7. Two compounds affected only males, 17 studies were negative, and in 13 studies with effects it was not possible to clearly determine the affected gender(s). Greater F0 dam weight was correlated with increased pup mass per litter; this relationship was strongest for the first litter, and weakest for the fifth litter. For both generations of treated females (F0 and F1), longer estrous cycles correlated with reduced numbers of pups; the relationship was stronger in F0 than in F1 females and was not seen in controls. Sperm parameters had different distributions in treated mice than in control mice. Fertility (total live pups/number of pairs cohabited) was reduced if there were > approximately 15% sperm abnormalities or if sperm motility (moving/not moving) was < approximately 37%. Both of these relationships appeared to have thresholds. Epididymal sperm count in treated animals, however, was linearly related to fertility, even within the control range, suggesting strongly that other factors are important. Using both treated and control data together, combining sperm count with motility could explain much (r = 0.77) of the variation in fertility; adding morphology did not significantly improve the correlation. The model was almost as strong using count and morphology, in which case adding motility did not strengthen the model. This analysis of these studies shows that while some endpoints (e.g., random-estrous-cycle-point ovary weight) correlate poorly with fertility, other necropsy endpoints (epididymal sperm count and motility, estrous cycle length, and testis and epididymal weights) can be useful (though not complete) surrogates of overall reproductive function. Indeed, over many studies, epididymal sperm count in treated animals correlates with fertility so well that even small reductions (approximately 20%) in count result in reduced fertility, suggesting that mice may be better models of human fertility than was previously believed.
The International Programme on Chemical Safety sponsored a collaborative study to evaluate the utility of neurobehavioral test methods for identifying neurotoxic chemicals. The protocol consisted of a functional observational battery and automated assessment of motor activity. The study involved four laboratories in the United States and four in Europe, each of which evaluated the dose- and time-related effects of seven prototypic chemicals following both single and 4-week repeated exposures. The protocol was designed to assess the general utility and reliability of neurobehavioral screening procedures in a diversity of testing situations. The results of chemical testing indicated that all participating laboratories generally could detect and characterize the effects of known neurotoxicants, despite some differences on specific endpoints. These data provide important information regarding the reliability and sensitivity of neurobehavioral screening methods over a range of laboratory conditions. The purpose of this workshop was to describe the background and study design of the collaborative effort, present the data (including comparison of results across laboratories), and discuss issues regarding the conduct and interpretation of these behavioral tests, as well as future directions for neurotoxicity screening.
Nitroaromatic musks, including musk ketone (MK; 2,6-dimethyl-3,5-dinitro-4-t-butylacetophenone), are chemicals used as perfume ingredients in household products, cosmetics, and toiletries. Musk xylene (MX; 1,3,5-trinitro-2-t-butylxylene), another nitromusk, is not genotoxic but has been reported to produce mouse liver tumors in a chronic bioassay. In addition, MX has been shown to both induce and inhibit mouse liver cytochrome P450 2B (CYP2B) isozymes. The ability of MX to inhibit CYP2B enzyme activity is attributable to inactivation of the enzyme by a specific amine metabolite. MK is structurally similar to MX, but lacks the nitro substitution that is reduced to the inactivating amine metabolite. Therefore, we hypothesized that MK would induce, but not inhibit, CYP2B isozymes. To test this hypothesis, and to evaluate the effects of MK on mouse liver cytochrome P450 enzymes, two sets of experiments were performed. To evaluate the ability of MK to induce cytochromes P450, mice were dosed daily by oral gavage at dosages ranging from 5 to 500 mg/ kg MK for 7 days. This treatment resulted in a pleiotropic response in mouse liver, including increased liver weight, increased total microsomal protein, and centrilobular hepatocellular hypertrophy. At the highest dose tested, MK caused a 28-fold increase in CYP2B enzyme activity and a small (approximately 2-fold) increase in both cytochromes P450 1A and 3A (CYP1A and CYP3A) enzyme activities over control levels. Protein and mRNA analyses confirmed the relative levels of induction for CYP2B, CYP1A, and CYP3A. In addition, the no-observable-effect level (NOEL) for CYP2B induction by MK was 20 mg/kg. To evaluate the ability of MK to inhibit phenobarbital-induced CYP2B activity, mice were given 500 ppm phenobarbital (PB) in the drinking water for 5 days to induce CYP2B isozymes, followed by a single equimolar (0.67 mmol/kg) oral gavage dose of either MK (198 mg/kg) or MX (200 mg/kg), and microsomes were prepared 18 h later. While MX inhibited more than 90% of the PB-induced CYP2B activity in the microsomes, MK caused only a small (about 20%) reduction in PB-induced CYP2B enzyme activity. These results indicate that, like MX. MK is a PB-type inducer of mouse liver CYP2B isozymes, but unlike MX, MK does not effectively inhibit PB-induced CYP2B enzyme activity.
Hydrazine belongs to a group of compounds for which there is evidence that the in vivo genotoxic effects become manifest only upon exposure to toxic dose levels. The present study was performed to investigate whether this phenomenon is also reflected in the pattern of DNA methylation. The induction of N7- and O6-methylguanine (MeGua) was studied in liver DNA of rats, 16 hr after treatment with various doses of hydrazine. After DNA isolation, the presence of N7-MeGua in DNA was assessed with an immunochemical method and with a physicochemical technique (HPLC with electrochemical detection). Application of these two methods resulted in almost identical patterns of dose-dependent induction of guanine N7-methylation in rats dosed orally with 0.1 to 10 mg hydrazine per kilogram of body weight, increasing from 1.1-1.3 to 39-45 N7-MeGua per 10(6) nucleotides. At lower dosages a constant adduct level was observed, equivalent to that in untreated rats (background level). The O6-MeGua level was analyzed by a combination of HPLC separation and competitive radioimmunoassay. A background level was observed for untreated rats and no increase was visible up to the 0.2 mg/kg dose group. After hydrazine doses from 0.2 to 10 mg/kg, O6-MeGua increased from 0.29 to 134 per 10(9) nucleotides. These data show that even at dosages below the maximum tolerated dose (0.6 mg/kg/day), for which carcinogenic effects have not been described, DNA adducts are formed. A comparison is made of the data obtained in this study with models that describe the mechanism of hydrazine-induced DNA methylation.
Determination of the ability of a medical device to interact with the immune system currently involves assessment of the immunogenic potential and biocompatibility of the device or an extract of the device. However, implants are often in the body for extended periods of time and/or are placed by a surgical procedure that in and of itself will generate an acute inflammatory response. This symposium discussed studies that have been performed to evaluate the immunogenicity of various devices consisting of several different compositions (i.e., silicone, metals, and latex) in contact with different anatomical sites, the ability of a device to modulate an inflammatory response generated by a surgical procedure or trauma, and the response of the body to a material left in place for extended periods of time. This symposium brought together scientists from many different disciplines to begin to identify and fill in the gaps in this area.
Previous studies have shown that acute, oral administration of malathion modulated the humoral immune response to T-cell-dependent antigen, mitogenic responses, macrophage function, and mast cell degranulation. While administration of malathion for 14 days did not affect the generation of an immune response to antigen, it was possible that macrophage and mast cell functions were affected. In this report, the effect of malathion administration for 14 days upon these parameters were assessed. This treatment regimen increased the respiratory burst capacity to a maximal level at a dose of 1 mg/kg/day or greater. The effect of oral administration of malathion for 14 days on the degranulation of mast cells in various organs (heart, skin, and small intestine) and peritoneal lavage fluid was also assessed. At doses of 1 mg/kg/day and above, the number of mast cells that was undegranulated decreased and the number that was severely degranulated increased. There was no change in mast cell integrity in biopsies from heart and skin, and in peritoneal fluid after 14-day administration of 0.1 mg/kg/day. However, the number of mast cells associated with the small intestine that had undergone degranulation was increased at this dose of malathion. These data indicate that repeated administration of malathion increased macrophage function at doses as low as 1 mg/kg/day and led to mast cell degranulation at doses as low as 0.1 mg/kg/day.
The risk from inhaled volatile organic compounds (VOCs) is presently assessed on the basis of lifetime exposure to average concentrations of the vapor. This strategy yields rational predictions of risk if the product of concentration (C) and the duration of exposure (t) yields constant effects on health (Haber's Rule). The validity of this assumption was evaluated by assessing the acute behavioral effects of inhaled trichloroethylene (TCE) vapor at various values ofCandt. Adult male Long–Evans rats (n= 11) were trained to perform a signal detection task in which a press on one lever produced food on trials containing a signal (a brief, unpredictable light flash); a press on a second lever produced food on trials lacking a signal. Response time (RT) and indices of sensitivity (SI) and bias (RI) derived from the theory of signal detection were calculated at three times during repeated daily 60-min tests conducted in air containing 0, 400, 800, 1200, 1600, 2000, or 2400 ppm TCE. Behavior remained stable during tests in air. In TCE, SI declined and RT increased as functions of bothCandt. RI was not affected by TCE. Effects on SI and RT were not predictable from theC × tproduct: both endpoints were more affected byCthan byt. To quantify the change in the effect of TCE across exposure times, concentration–effect relationships for inhaled TCE on SI and RT were modeled with cubic polynomial functions at each of the three exposure durations. Concentrations of inhaled TCE associated with preselected changes in SI and RT were then estimated for each animal from these functions. Criterion concentrations, SI0.1and RT100, were defined as the concentration of TCE associated with a 0.1-unit decrease in SI or a 100-msec increase in RT, respectively. Both SI0.1and RT100increased as exposure duration decreased, but did so more slowly than would be predicted by Haber's Rule. This pattern indicates that application of Haber's Rule overestimates the concentration of inhaled TCE associated with changes in signal detection and thus underestimates the risk of behavior change from short-term exposures to TCE. On the other hand, the fact that SI0.1and RT100did increase with shorter exposure times indicates that the converse assumption, that the toxicity of inhaled TCE is independent of the duration of exposure, yields an overly conservative estimate of risk.
The extent to which cardiorespiratory infirmity and other sublethal effects of saxitoxin (STX) and tetrodotoxin (TTX) can be reversed by 4-aminopyridine (4-AP) was investigated in guinea pigs chronically instrumented for the concurrent electrophysiological recordings of electrocorticogram (ECoG), diaphragmatic electromyogram (DEMG), Lead II electrocardiogram, and neck skeletal muscle electromyogram. Animals were intoxicated with either STX or TTX (2 and 3 μg/kg, im) to produce a state of progressive cardiorespiratory depression (depicted by decreasing DEMG amplitude, bradypnea, and bradycardia). At the point where cardiorespiratory performance was most seriously compromised (≈30 min posttoxin), 4-AP (1 or 2 mg/kg, im) was administered. The therapeutic effect of 4-AP was striking in that, within minutes, the toxin-induced diaphragmatic blockade, bradypnea, bradycardia, and depressed cortical activity were all restored to a level either comparable to, or surpassing, that of control. The optimal 4-AP dose level was determined to be 2 mg/kg (im) based on analyses of cardiorespiratory activity profiles throughout the course of intoxication and 4-AP treatment. At the dose levels (either 1 or 2 mg/kg) used to restore ventilatory function and cardiovascular performance, 4-AP produced no sign of seizures and convulsions. Although less serious secondary effects such as cortical excitant/arousal effect (indicated by ECoG power spectral analysis) and transient periods of skeletal muscle fasciculation were observed, these events were of minor concern particularly in view of the remarkable therapeutic effects of 4-AP.
A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market. In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r = 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NOx, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by these assays.
The potent carcinogenicity of dibenzo[a,l]pyrene in mouse skin is associated with an inflammation unique among polycyclic aromatic hydrocarbons and expressed as erythema. The time course of erythema and the associated histological events in the skin of female SENCAR mice were determined after a single application of 6.25–200 nmol dibenzo[a,l]pyrene or selected metabolites. Dibenzo[a,l]pyrene and dibenzo[a,l]pyrene-11,12-dihydrodiol, precursor to the bay-region diol epoxide, induced an erythema first present 5–6 days after treatment Dibenzo[a,l]pyrene-8,9-dihy-drodiol and other dibenzo[a,l]pyrene metabolites, however, did not induce erythema. These findings suggest a central role for the bay-region diol epoxide in the induction of the observed inflammation. The intensity and duration of erythema were dose-dependent, whereas the delayed appearance of erythema was constant and dose-independent. These results suggest induction of an immune hypersensitivity by dibenzo[a,l]pyrene and its 11,12-dihydrodiol. Histological changes in the skin were consistent with a contact hypersensitivity reaction and included, in association with erythema, epidermal hyperplasia and the presence of mononuclear leukocytes in the dermis. Animals were tested for dibenzo[a,l]-pyrene-induced contact hypersensitivity. Female SENCAR mice were treated with a single dermal application of dibenzo[a,l]pyrene or 7,12-dimethylbenz[a]anthracene. Five days later, the animals were challenged with a single application of dibenzo[a,l]pyrene or 7,12-dimethylbenz[a]anthracene to the ear pinna. Ear swelling exhibited features of a contact hypersensitivity reaction, including (1) delayed appearance after challenge, (2) noninducibility in animals not previously exposed to chemical sensitizer, and (3) chemical specificity. The results suggest that dibenzo[a,l]pyrene induces, via its bay-region diol epoxide, a contact hypersensitivity reaction that may promote tumor development and thereby enhance carcinogenic potency.
The side effects of recombinant human interleukin-2 (rhIL-2) in mice are considered to be mediated by lymphokine-activated killer (LAK) cells that have cytoplasmic granules and infiltrate into target organs. In cloned cytotoxic lymphocytes (CLs) including LAK cells in vitro, granule exocytosis is one of the mechanisms in target cell lysis, and hemolytic and BLT-serine esterase (BLT-SE) activities detected in the granules are important to the cytotoxicity of the CLs in vitro. However, no information is available on these activities of LAK granules in vivo in mice showing the side effects of rhIL-2. Therefore, the present study was designed to isolate the LAK granules from the homogenate of LAK cell-rich subcutaneous tissue in mice treated with continuous infusion of a dose of rhIL-2 that induces known toxicity. High activities in hemolysis and BLT-SE were detected in Percoll fractions from the tissue homogenate. Electron microscopic observation of these fractions revealed almost only LAK granules. These results indicate that hemolytic and BLT-SE activities are detectable in the LAK granules in vivo, suggesting that the cells have potency for cytotoxicity via their granules and are involved in the toxicity of rhIL-2 in mice. In addition, perforin, which has been identified as a hemolytic protein in CL granules in vitro, was not detected by immunoblotting in the granule fraction of the LAK cells in vivo, indicating that the hemolytic activity of the LAK granules in vivo is not due to perforin.
Peroxisome proliferators cause liver cell proliferation in addition to other pleiotropic effects such as peroxisome proliferation and induction of certain peroxisomal and cytosolic enzymes in liver. Since dexamethasone has been shown to inhibit mitogen-induced liver cell hyperplasia, we examined whether dexamethasone inhibits only cell proliferation without affecting peroxisome proliferation induced by peroxisome proliferators such as ciprofibrate. Livers of rats fed a diet containing ciprofibrate (0.025%) with or without added dexamethasone (0.5 mg or 1 mg/kg diet) for 1 week were evaluated for hepatocyte proliferation and peroxisome proliferation. Dexamethasone administration resulted in abrogation of ciprofibrate-induced cell proliferation as shown by bromodeoxyuridine (BrdU) labeling and mitoses counts. The hepatocyte proliferative index measured after administration of a single dose of BrdU was 18.3 +/- 1.1 and 2.3 +/- 0.7% (p < 0.01) in ciprofibrate and ciprofibrate + dexamethasone treated rats, respectively. With multiple injections of BrdU (daily injections for 7 days) the proliferative index was 225 +/- 10 and 183 +/- 2% (p < 0.02), respectively, in these two groups. Interestingly, whereas the levels of peroxisome proliferator-induced Mr 80,000 polypeptide and catalase and peroxisomal bifunctional enzyme, and the corresponding mRNAs and peroxisome volume density were unaffected. These results show that dexamethasone selectively inhibits only cell proliferation without inhibiting the peroxisome proliferation caused by ciprofibrate. This model should be useful for examining the role of cell proliferation versus oxidative stress in peroxisome proliferator-induced hepatocarcinogenesis.
Many marketed pharmaceuticals are known to cause idiosyncratic agranulocytosis in humans. Similarly prinomide, an antiinflammatory drug, was associated with a low incidence of agranulocytosis (<0.3%) in clinical trials, even though chronic toxicity studies in rodents and primates showed no evidence of agranulocytosis with either prinomide or its parahydroxy metabolite, CGS 12094. To investigate mechanisms for this human specific toxicity, experiments were conducted to study the metabolism of prinomide and CGS 12094 by myeloperoxidase (MPO), a major enzyme of neutrophils and leukocyte progenitor cells. Although prinomide was not metabolized by human MPO, CGS 12094 was rapidly metabolized (>90%; 2 min); this reaction was dependent on H2O2 and MPO and was inhibited by azide. During the MPO-catalyzed metabolism of CGS 12094, reactive intermediates that irreversibly bound to protein and cysteine were generated. One of the reactive metabolites generated was identified by mass spectroscopy and trapping with cysteine as 1,4-benzoquinone, a compound implicated in the myelotoxicity associated with benzene. Thus during conditions which lead to elevated levels of H2O2 (e.g., active inflammation), CGS 12094 is rapidly metabolized by MPO to reactive intermediates that may be related to prinomide-induced agranulocytosis.
Long-term inhalation exposure of benzene has been shown to cause hematotoxicity and an increased incidence of acute myelogenous leukemia in humans. The progression of benzene-induced hematotoxicity and the features of the toxicity that may play a major role in the leukemogenesis are not known. We report the hematological consequences of benzene inhalation in B6C3F1 mice exposed to 1, 5, 10, 100, and 200 ppm benzene for 6 hr/day, 5 days/week for 1, 2, 4, or 8 weeks and a recovery group. There were no significant effects on hematopoietic parameters from exposure to 10 ppm benzene or less. Exposure of mice to 100 and 200 ppm benzene reduced the number of total bone marrow cells, progenitor cells, differentiating hematopoietic cells, and most blood parameters. Replication of primitive progenitor cells in the bone marrow was increased during the exposure period as a compensation for the cytotoxicity induced by 100 and 200 ppm benzene. In mice exposed to 200 ppm benzene, the primitive progenitor cells maintained an increased percentage of cells in S-phase through 25 days of recovery compared with controls. The increased replication of primitive progenitor cells in concert with the reported genotoxicity induced by benzene provides the components necessary for producing an increased incidence of lymphoma in mice. Furthermore, we propose this mode of action as a biologically plausible mechanism for benzene-induced leukemia in humans exposed to high concentrations of benzene.
Rats are more sensitive to ozone-induced pulmonary inflammation and damage during late pregnancy and throughout lactation than in pre- or early pregnancy or post lactation. This window of sensitivity coincides with a period of elevated levels of pituitaryderived prolactin or placental lactogen. In this study, we investigated the hypothesis that prolactin exerts an enhancing effect on ozone-induced pulmonary inflammation and damage, thus presenting a plausible explanation for the sensitivity profile observed in rats. Hyperprolactinemia was achieved by using rats with subcutaneous tumors that were derived from the MMQ tumor model previously described by Adler and co-workers (Adler, R. A., Krieg, R. J., Farrell, M. E., Deiss, W. P., and MacLeod, R. M., Metabolism 40, 286–291, 1991). A variant of the MMQ tumor, the MMQr tumor, which appeared spontaneously from a single passage of MMQ tumor tissue, produced elevated levels of corticosterone in addition to high levels of prolactin. These two subcutaneous tumors had markedly different effects on adrenal, thymus, and spleen weights because of the different hormonal milieu they generated. There was also a significant difference between MMQ- and MMQr-bearing rats in their inflammatory response to acute ozone exposure as assessed by polymorphonuclear leukocytes (PMNs) in the airways. Rats with MMQ tumors were not significantly different from non-tumor-bearing controls in their baseline level of airway PMNs and PMN inflammation following ozone exposure, whereas MMQr-bearing rats had significantly elevated baseline PMNs in their airways and a greater PMN response to inhaled ozone. The hormonal milieu and elevated PMNs in the airways of both unexposed and ozone-exposed rats with MMQr tumors were similar to levels observed in lactating rats. The role of corticosterone in pulmonary inflammation in this model was investigated further by treating MMQ tumor-bearing rats with dexamethasone. Dexamethasone was effective in producing changes in organ weights similar to those observed in MMQr rats, but did not elicit higher airway PMN concentrations in unexposed rats as observed in the MMQr rats. We conclude that in this animal model prolactin did not significantly elevate airway PMN inflammation induced by ozone, and supplementation with exogenous glucocorticoid did not duplicate the endogenous airway PMNs numbers observed in MMQr-bearing rats or lactating rats.
In vitro assessment of human lymphocyte viability by trypan blue dye exclusion in the presence of an external metabolizing system (microsomes plus NADPH) has been shown to be a useful method in assessing predisposition to idiopathic toxicity in response to various anticonvulsant drugs. The trypan blue method, however, is labor intensive, is time consuming, is prone to human error, is not suitable for high-volume toxicity screening, and excludes autolysed cells. The objective of this study was to develop a rapid, high-capacity, objective, and easy in vitro cytotoxicity method for the detection of metabolism-dependent cytotoxicity of a test chemical. The in vitro system uses an external metabolizing system (rabbit microsomes) in conjunction with isolated human lymphocytes as the target cells. Cellular toxicity was determined by assessing plasma membrane integrity using a membrane-impermeant fluorescent nucleic acid dye (YO-PRO-1) and a multiwell plate scanner for fluorescence. Using this system, cells incubated with either acetaminophen (1500 micrograms/ml), carbamazepine (62.5 microM), phenytoin (62.5 microM), or phenobarbital (62.5 microM) showed net increases in percentage cell death of 31 +/- 5, 11 +/- 4, 0 +/- 3, and 2 +/- 3, respectively. A metabolism-dependent concentration-response was observed for valproic acid-induced cytotoxicity, which approached a plateau at a concentration of 4000 micrograms/ml with a net percentage cell death of 31 +/- 4. This technique resolves various technical difficulties inherent in viability determinations by the trypan blue exclusion method. The YO-PRO-1 method also may be useful in a clinical setting for the assessment of patients with a genetically determined susceptibility to certain drugs and for identifying the responsible drug in patients with idiopathic toxicity undergoing multiple-drug therapy.
Previous research showed that acute exposure to dichloromethane (DCM) produced a selective reduction in peak N30 of flash evoked potentials (FEPs) in rats. In contrast, acute exposures to p-xylene or toluene selectively reduced FEP peak N160. The present experiments compared the effects of DCM (log P = 1.25; oil:water partition coefficient), 1,3-dichloropropane (DCP; log P = 2.00), and 1,2-dichlorobenzene (DCB; log P = 3.38) on FEPs recorded from adult Long-Evans rats. Before administration of test compounds, FEPs were recorded for five daily sessions to develop FEP peak N160. Test compounds were dissolved in corn oil and administered i.p. at doses based on proportions of their LD50 values. The doses were: DCM, 0, 57.5, 115, 230, or 460 mg/kg; DCP, 0, 86, 172, 343, or 686 mg/kg; and DCB, 0, 53, 105, 210, or 420 mg/kg. Testing times after dosing varied among compounds and were based on pilot studies to measure both the times of peak effect and recovery. Each solvent produced significant changes in the latency and amplitude of multiple components of the FEP waveforms. However, the predominant effect of DCM was to reduce the amplitude of peak N30 (ED50 = 326.3 mg/kg), that of DCP was to reduce both peaks N30 (ED50 = 231.0 mg/kg) and N160 (ED50 = 136.8 mg/kg), and that of DCB was to reduce peak N160 (ED50 = 151.6 mg/kg). There was no consistent relationship between log P values and the potency of the compounds to alter FEP peaks N30 and N160. The results suggest that organic solvents have multiple acute effects on the function of the central nervous system, which are not predictable solely by the compound's lipid solubility.
The mesothelial lining is a target for the fibrotic and carcinogenic effects of mineral fibers. Fiber geometry, dimensions, chemical composition, surface reactivity, and biopersistence at the target tissue have been proposed to contribute to these toxic endpoints. We established a dose-response relationship between the number of fibers delivered to the parietal peritoneal lining, inflammation, and mesothelial cell proliferation induced by intraperitoneal injection of crocidolite asbestos fibers in mice. Persistence of these inflammatory and proliferative responses depended on persistence of fibers at the target tissue. Intraperitoneal injection of wollastonite fibers induced an early inflammatory and proliferative response that subsided after 21 days. Approximately 50% of wollastonite fibers were recovered by bleach digestion after 21 days and only 2% were recovered after 6 months. In contrast, the number of fibers recovered from tissue digests had not declined 6 months after injection of crocidolite asbestos. These results support the hypothesis that biopersistent fibers cause persistent inflammation and chronic mesothelial cell proliferation.
