Induction of c-jun and TGF-beta 1 in Fischer 344 rats during amiodarone-induced pulmonary fibrosis.

Amiodarone (AM) is an antidysrhythmic agent with a propensity to cause pulmonary toxicity, including potentially fatal fibrosis. In the present study, the potential roles of c-Jun and transforming growth factor (TGF)-beta 1 in AM-induced inflammation and fibrogenesis were examined after intratracheal administration of AM (1.83 micromol/day on days 0 and 2) or an equivalent volume (0.4 ml) of distilled water to male Fischer 344 rats. Northern and immunoblot analyses demonstrated that lung TGF-beta 1 (mRNA and protein) expression was increased 1.5- to 1.8-fold relative to control during the early inflammation period and 1 day, 1 wk, and 2 wk post-AM treatment. Lung c-Jun protein expression was increased concomitantly with evidence of AM-induced fibrosis; at 5 wk post-AM treatment, c-Jun protein was increased 3.3-fold relative to control. The results indicate a role for induction of c-jun and TGF-beta 1 expression in the development of AM-induced pulmonary fibrosis in the Fischer 344 rat and provide potential targets for therapeutic intervention.

Effects of vitamin E on cytotoxicity of amiodarone and N-desethylamiodarone in isolated hamster lung cells.

Amiodarone (AM) is a potent and efficacious antidysrhythmic agent that can cause potentially life-threatening pulmonary fibrosis. Vitamin E has been demonstrated to decrease AM-induced pulmonary fibrosis in vivo in hamsters. In the present in vitro study, we investigated the effects of vitamin E on cell death induced by AM and its primary metabolite, N-desethylamiodarone (DEA), in freshly isolated hamster lung cells. Following incubation for 24 or 36 h, 300 microM vitamin E decreased (P<0.05) 100 microM AM-induced cytotoxicity (0.5% trypan blue uptake) in alveolar macrophages by 11.7+/-3% or 21.4+/-12%, respectively, but did not decrease cytotoxicity in fractions enriched with alveolar type II cells or non-ciliated bronchiolar epithelial (Clara cells) or in isolated unseparated cells (cell digest). Vitamin E had no effect on 50 microM DEA-induced cytotoxicity. Vitamin E did not alter cellular levels of AM or DEA in any cell fraction. Lipid peroxidation (assessed by isoprostane formation) was increased (P<0.05) in cell digest, alveolar type II cell and Clara cell enriched fractions incubated with 500 microM carbon tetrachloride (CCl(4)) for 4 h but not in enriched fractions of cells exposed to 100 microM AM or 50 microM DEA. No AM-induced loss of viability was observed at this time point, but DEA decreased (P<0.05) Clara cell viability by approximately 25%. These results demonstrate cell type selective protection against AM-induced cytotoxicity by vitamin E, and suggest that lipid peroxidation does not initiate AM- or DEA-induced cytotoxicity in isolated hamster lung cells.

Disruption of mitochondrial function and cellular ATP levels by amiodarone and N-desethylamiodarone in initiation of amiodarone-induced pulmonary cytotoxicity.

Amiodarone (AM), a potent antidysrhythmic agent, can cause potentially life-threatening pulmonary fibrosis. In the present investigation of mechanisms of initiation of AM lung toxicity, we found that 100 microM AM decreased mitochondrial membrane potential in intact hamster lung alveolar macrophages and preparations enriched in isolated alveolar type II cells and nonciliated bronchiolar epithelial (Clara) cells, following 2 h of incubation. This was followed by a drop in cellular ATP content (by 32--77%) at 4 to 6 h, and 30 to 55% loss of viability at 24 h. Supplementation of incubation media with 5.0 mM glucose or 2.0 mM niacin did not reduce AM-induced ATP depletion or cell death in macrophages, and the mitochondrial permeability transition inhibitor cyclosporin A (1.0 microM) did not affect AM cytotoxicity. At 50 microM, the AM metabolite N-desethylamiodarone (DEA) produced effects similar to those of AM, but more rapidly and extensively, with the Clara cell-enriched preparation being particularly susceptible. In isolated whole lung mitochondria, DEA was accumulated to a greater extent than AM. Both AM and DEA inhibited complex I- and complex II-supported respiration, but DEA inhibited complex II to a greater degree than AM. These results demonstrate that AM and DEA disrupt mitochondrial membrane potential prior to ATP depletion and subsequent lung cell death, that DEA is more potent than AM, and that the mitochondrial permeability transition is not involved in mitochondrial perturbation by AM. This suggests that AM- and DEA-induced perturbations of mitochondrial function may initiate AM-induced pulmonary toxicity.

The role of mitochondrial injury in bromobenzene and furosemide induced hepatotoxicity.

Bromobenzene (BB) and furosemide (FS) are two hepatotoxicants whose bioactivation to reactive intermediates is crucial to the development of liver injury. However, the events which lead to hepatocellular toxicity following metabolite formation and covalent binding to cellular macromolecules remain unknown. The present study was undertaken to investigate the effect of administered BB and FS on mitochondrial total glutathione (GSH+GSSG, henceforth referred to as glutathione) content and respiratory function as potential initiating mechanisms of the hepatotoxicity of these compounds in the mouse. Bromobenzene (2 g/kg i.p.) significantly decreased mitochondrial glutathione to 48% of control at 3 h post administration, and to 41% at 4 h. This decrease in mitochondrial glutathione was subsequent to a significant decrease in cytosolic glutathione to 64 and 28% of control at 1 and 2 h, respectively. Oxygen consumption supported by complex I (glutamate-supported) of the respiratory chain was not inhibited by BB until 4 h, where state 3 (active) respiration was reduced to 16% of control. This resulted in a decreased respiratory control ratio (RCR) for complex I-supported respiration. Complex II (succinate)-supported state 3 and state 4 respiration were unaffected by BB until 4 h, at which time they were reduced to 57 and 48% of control, respectively. However, the similar reductions in state 3 and state 4 respiratory rates did not alter the corresponding RCR for complex II. Overt hepatic injury was detected at 4 h, with plasma alanine aminotransferase (ALT) activity increasing significantly at this time point. In contrast to the effects of BB, FS administration (400 mg/kg i.p.) did not alter mitochondrial or cytosolic glutathione, and had no effect on respiration supported by complex I or II for up to 5 h following dosing. However, ALT activity was significantly increased 5 h following FS administration. These results suggest that inhibition of mitochondrial respiratory function coinciding with a decrease in mitochondrial glutathione content may be crucial to the initiation of BB-induced hepatotoxicity, while such events are not required for the initiation of FS-induced hepatotoxicity.

Effects of an environmental anti-androgen on erectile function in an animal penile erection model.

PURPOSE: Erectile function is testosterone dependent. For example, interference with either the levels or receptor binding of this steroid hormone may induce erectile dysfunction. Several environmental contaminants can interfere with the actions of endogenous hormones and have been termed 'endocrine disrupters.' p,p-DDE, a prominent and persistent metabolite of the insecticide DDT, has been shown to be an androgen receptor antagonist. The objective was to determine whether endocrine disrupters, as exemplified by p,p-DDE, are factors in the etiology of erectile dysfunction. MATERIALS AND METHODS: Using the established rat model of apomorphine-induced (80 microg./kg, s.c.) erections we assessed the dose-response effects of p,p-DDE in comparison to the known androgen receptor antagonist flutamide in acute (0.5 to 12 hours) and short-term (up to 8 weeks) experiments in both intact (Study 1) and castrated (Study 2) rats. As a follow up (Study 3), castrated rats treated with p,p-DDE were given increasing doses of testosterone (0.48 to 2.4 mg./kg., i.p.), eight weeks after p,p-DDE administration, to assess reversibility of p,p-DDE effect. RESULTS: A single dose of flutamide (50 mg./kg., i.p.) was found to significantly decrease apomorphine-induced erections to less than 50% over 12 hours following flutamide administration with recovery of erectile response within 48 hours. In comparison, a single dose of p,p-DDE (500 mg./kg., i.p.) decreased apomorphine-induced erections for at least two weeks (1.15+/-0.3 versus 2.5+/-1.1). Castration significantly decreased apomorphine-induced erections to approximately 0.5 erections/30 minutes. Flutamide (50 mg./kg.; i.p.) or p,p-DDE (50 mg./kg.; i.p.) did not further suppress the apomorphine erections in castrated rats. Testosterone supplementation (480 microg./kg; s.c.) in vehicle treated castrated rats recovered erectile response to pre-castrated levels, whereas p,p-DDE treated castrated rats required 4 times the dose of testosterone (2 mg./kg.; s.c.) given to vehicle treated rats to recover erections. CONCLUSIONS: The endocrine disrupter p,p-DDE can markedly interfere with erectile function and demonstrates persistence after a single dose. This supports our novel concept that environmental hormones may cause erectile dysfunction.

Effects of dietary vitamin E supplementation on pulmonary morphology and collagen deposition in amiodarone- and vehicle-treated hamsters.

Amiodarone (AM) is a potent antidysrhythmic agent that is limited in clinical use by its adverse effects, including potentially life-threatening AM-induced pulmonary toxicity (AIPT). The present study tested the ability of dietary supplementation with vitamin E (500 IU d,1-alpha-tocopherol acetate/kg chow) to protect against pulmonary damage following intratracheal administration of AM (1.83 micromol) to the male golden Syrian hamster. At 21 days post-dosing, animals treated with AM had increased lung hydroxyproline content and histological disease index values compared to control (P < 0.05), which were indicative of fibrosis. Dietary vitamin E supplementation for 6 weeks resulted in a 234% increase in lung vitamin E content at the time of AM dosing, and maintenance on the diet prevented AM-induced elevation of hydroxyproline content and disease index 21 days post-dosing. Dietary vitamin E supplementation also decreased hydroxyproline content and disease index values in hamsters treated intratracheally with distilled water, the AM vehicle. These results demonstrate a protective role for vitamin E in an in vivo model of AIPT, and suggest that this antioxidant may have non-specific antifibrotic effects in the lung.

Amiodarone-induced disruption of hamster lung and liver mitochondrial function: lack of association with thiobarbituric acid-reactive substance production.

Amiodarone (AM) is an efficacious antidysrhythmic agent that is limited clinically by numerous adverse effects. Of greatest concern is AM-induced pulmonary toxicity (AIPT) due to the potential for mortality. Mitochondrial alterations and free radicals have been implicated in the etiology of AM-induced toxicities, including AIPT. Isolated hamster lung and liver mitochondria were assessed for AM-induced effects on respiration, membrane potential, and lipid peroxidation. AM (50-400 microM) stimulated state 4 (resting) respiration at complexes I and II of tightly coupled lung mitochondria, with higher concentrations (200 and 400 microM) resulting in a subsequent inhibition. This biphasic effect of AM (200 microM) was also observed with isolated liver mitochondria. Only inhibition of respiration was observed with AM (50-400 microM) in less tightly coupled lung mitochondria. Based on safranine fluorescence, 200 microM AM decreased lung mitochondrial membrane potential (p < 0.05), while a concentration-dependent (50-200 microM) decrease of membrane potential was observed with liver mitochondria exposed to AM (p < 0.05). Formation of thiobarbituric acid-reactive substances (TBARS) was not altered by AM (50-400 microM) in incubations lasting up to 1 h. These results indicate that lipid peroxidation, as indicated by levels of TBARS, does not play a role in AM-induced alterations in mitochondrial respiration and membrane potential.

Differential susceptibilities of isolated hamster lung cell types to amiodarone toxicity.

Treatment of cardiac dysrhythmias with the iodinated benzofuran derivative amiodarone (AM) is limited by pulmonary toxicity. The susceptibilities of different lung cell types of male Golden Syrian hamsters to AM-induced cytotoxicity were investigated in vitro. Bronchoalveolar lavage and protease digestion to release cells, followed by centrifugal elutriation and density gradient centrifugation, resulted in preparations enriched with alveolar macrophages (98%), alveolar type II cells (75-85%), and nonciliated bronchiolar epithelial (Clara) cells (35-50%). Alveolar type II cell and Clara cell preparations demonstrated decreased viability (by 0.5% trypan blue dye exclusion) when incubated with 50 microM AM for 36 h, and all AM-treated cell preparations demonstrated decreased viability when incubated with 100 or 200 microM AM. Based on a viability index ((viability of AM-treated cells/viability of controls) x 100%), the Clara cell fraction was significantly (p<0.05) more susceptible than all of the other cell types to 50 microM AM. However, AM cytotoxicity was greatest (p<0.05) in alveolar macrophages following incubation with 100 or 200 microM AM. There was no difference between any of the enriched cell preparations in the amount of drug accumulated following 24 h of incubation with 50 microM AM, whereas alveolar macrophages accumulated the most drug during incubation with 100 microM AM. Thus, the most susceptible cell type was dependent on AM concentration. AM-induced cytotoxicity in specific cell types may initiate processes leading to inflammation and pulmonary fibrosis.

The effects of methylmercury on endogenous dopamine efflux from mouse striatal slices.

The present study investigated the effects of in vitro methylmercury (MeHg) exposure on endogenous dopamine (DA) efflux from mouse striatal slices. MeHg produced a concentration-dependent increase in the spontaneous efflux of DA which was independent of the availability of Ca2+ in the superfusion medium. The Ca(2+)-dependent K(+)-evoked release of DA was significantly enhanced by 50 and 100 microM MeHg. This increase could not be solely accounted for by the MeHg-induced increased in spontaneous DA efflux. The K(+)-stimulated efflux of DA was enhanced by MeHg in both the presence and absence of Ca2+ in the superfusion medium, suggesting that under depolarizing conditions, DA efflux induced by MeHg has a Ca(2+)-independent component. The alterations in DA efflux occurred at concentrations of MeHg previously found in the CNS of animals exhibiting symptoms of MeHg intoxication suggesting that alterations in DA neurotransmission in the striatum may contribute to the symptoms of MeHg toxicity.

Bromobenzene and furosemide hepatotoxicity: alterations in glutathione, protein thiols, and calcium.

The nature of the events whereby the reactive intermediates resulting from the bioactivation of bromobenzene and furosemide induce hepatotoxicity is unknown. To examine a role for disturbances in intracellular calcium homeostasis, secondary to a depletion in cellular reduced glutathione (GSH) and reduced protein thiols (PSHs), isolated mouse hepatocytes were exposed to cytotoxic concentrations of bromobenzene or furosemide. Cytosolic calcium concentration, as well as thiol status, was determined. The incubation of hepatocytes with 3.0 mM bromobenzene, and subsequent additions (1.2 mM) of the agent every hour, resulted in significant GSH depletion. The loss of plasma membrane integrity at 1.5 h preceded both a rise in the cytosolic Ca2+ concentration and depletion of total PSH content. Furosemide (1.0 mM) produced a 70% depletion in cellular GSH content in isolated hepatocytes. The initiation of cell damage occurred concurrently with both a rise in the cytosolic Ca2+ concentration and a depletion of total PSH content 4 h following furosemide addition. Since the increase in cytosolic Ca2+ did not precede cytotoxicity, these results do not support an initiating role for Ca2+ deregulation in bromobenzene and furosemide hepatotoxicities. In addition, depletion of PSH content did not correlate with bromobenzene- or furosemide-induced cytotoxicity.

Effects of N-acetylcysteine and dithiothreitol on glutathione and protein thiol replenishment during acetaminophen-induced toxicity in isolated mouse hepatocytes.

Isolated mouse hepatocytes were incubated with 1.0 mM acetaminophen (AA) for 1.5 h to initiate glutathione (GSH) and protein thiol (PSH) depletion and cell injury. Cells were subsequently washed to remove non-covalently bound AA and resuspended in medium containing N-acetylcysteine (NAC, 2.0 mM) or dithiothreitol (DTT, 1.5 mM). The effects of these agents on the replenishment of GSH and total PSH content were related to the development of cytotoxicity. When cells exposed to AA were resuspended in medium containing NAC or DTT, both agents replenished GSH and total PSH content to levels observed in untreated cells but only DTT was able to attenuate cytotoxicity. Addition of the GSH synthesis inhibitor, buthionine sulfoximine (BSO, 1.0 mM, 1.5 h), to cells in incubation medium containing AA, enhanced GSH and total PSH depletion and potentiated cytotoxicity. Resuspension of these cells in medium containing NAC did not alter the potentiating effects of BSO; GSH and PSH levels were not replenished and no cytoprotective effects were observed. However, when cells exposed to AA and BSO were resuspended in medium containing DTT, PSH content was replenished but GSH levels were not restored. In addition, DTT was able to delay the development of cytotoxicity. It appears that DTT, unlike NAC, has a GSH-independent mechanism of PSH replenishment. These observations suggest that while replenishment of GSH and total PSH content does not result in cytoprotection, the regeneration of critical PSH by DTT may play an important role in the maintenance of proper cell structure and/or function.

Inhibition of mitochondrial respiration in vivo is an early event in acetaminophen-induced hepatotoxicity.

Morphological changes in mitochondria are observed early in the course of acetaminophen (AA)-induced hepatotoxicity, and mitochondrial dysfunction has been observed both in vivo and in vitro following exposure to AA. This study examined the early effects of AA exposure in vivo on mitochondrial respiration and evaluated the effectiveness of N-acetyl-L-cysteine (NAC) in protecting against respiratory dysfunction. Mitochondria were isolated from the livers of fasted, male CD-1 mice 0, 0.5, 1, 1.5 or 2 h after administration of a hepatotoxic dose of AA (750 mg/kg). Glutamate- and succinate-supported mitochondrial respiration were subsequently assessed by polarographic measurement of state 3 (ADP-stimulated) and state 4 (resting) rates of oxygen consumption and determination of the corresponding respiratory control ratios (RCR: state 3/state 4) and ADP:O ratios. Hepatotoxicity was assessed histologically and by measuring plasma alanine aminotransferase (ALT) activity. The earliest sign of mitochondrial dysfunction observed in this study was a significant decrease in the ADP:O ratio for the oxidation of glutamate 1 h post-dosing. At 1.5 and 2 h post-dosing the RCRs for both glutamate- and succinate-supported respiration were significantly decreased. All of the respiratory parameters measured in this study were significantly decreased, with the exception of succinate-supported state 4 respiration which was significantly increased, 2 h after AA administration. Thus, inhibition of mitochondrial respiration preceded overt hepatic necrosis, indicated by an elevation of ALT activity, which was not observed until 3 and 4 h post-dosing. In addition, mitochondrial respiratory dysfunction correlated with morphological alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular calcium disruption as a secondary event in acetaminophen-induced hepatotoxicity.

To examine a role for disturbances in intracellular calcium homeostasis in acetaminophen-induced hepatotoxicity, freshly isolated mouse hepatocytes were incubated with 1.0 mM acetaminophen for 1.5 h to allow for covalent binding and initiation of cell damage. The hepatocytes were then washed and the cells incubated in fresh medium containing either 2.0 mM N-acetylcysteine or 1.5 mM dithiothreitol for the duration of a 4-h incubation period. These agents were used as tools in the elucidation of the biochemical events responsible for acetaminophen-induced cell necrosis. The reduced protein sulfhydryl content, cytosolic [Ca2+], and plasma membrane integrity were quantitated. Acetaminophen produced protein sulfhydryl depletion, an increased cytosolic [Ca2+], and cell injury; however, cytotoxicity preceded the increase in [Ca2+]. Both N-acetylcysteine and dithiothreitol restored the acetaminophen-induced protein sulfhydryl loss. Dithiothreitol prevented both further cell injury and an increase in the cytosolic [Ca2+]. However, cell death and a subsequent increase in cytosolic [Ca2+] proceeded unabated following N-acetylcysteine addition. Although both agents restored protein sulfhydryl content, in view of their contrasting ultimate effects on cell viability the role of reduced protein sulfhydryl depletion in acetaminophen-induced hepatic injury requires further investigation. The increase in cytosolic [Ca2+] with acetaminophen alone and with subsequent N-acetylcysteine addition was determined to be a secondary event in cell injury because cytotoxicity occurred by 1.5 h; however, the increase in cytosolic [Ca2+] was not observed until 2.5 h. Additional evidence for changes in cytosolic [Ca2+] as a secondary event was obtained by incubating the hepatocytes with acetaminophen in the presence of fura 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative methods of selecting rat hepatocellular nodules resistant to 2-acetylaminofluorene.

Dietary 2-acetylaminofluorene (2-AAF) coupled with a stimulus for cell proliferation such as a 2/3 partial hepatectomy (PH) or a necrotizing dose of carbon tetrachloride is frequently employed to generate nodules of resistant ("initiated") rat hepatocytes. This regimen is a useful model for experimental analysis of alterations in hepatocytes during carcinogenesis, and also as an assay for initiation by various carcinogens. Because of the decreasing availability of carcinogen-containing diets from commercial sources, we have developed alternative methods of 2-AAF administration to generate nodules in rats initiated with N-nitrosodiethylamine. This study compared the nodule-selecting and cancer-promoting efficacy of 2-AAF administered by the Solt-Farber procedure (0.02% in diet for 2 weeks) with 2-AAF administered by gavage, as a suspension in 1% aqueous carboxymethyl-cellulose (CMC). Three or 4 daily administrations of 2-AAF by gavage (20 mg/kg/day) followed by PH on day 4 were equivalent to the dietary regimen in generating early resistant nodules, late persistent nodules and hepatocellular carcinomas. These regimens were similar to the dietary regimen of 2-AAF in inhibiting virtually all normal hepatocyte proliferation. These regimens permit control over the duration and level of 2-AAF exposure and the resulting size of selected nodules.

Metabolic activation of 1,1-dichloroethylene by mouse lung and liver microsomes.

1,1-Dichloroethylene (1,1-DCE) causes lung and liver necrosis in mice. Covalent binding of [14C]1,1-DCE to isolated lung and liver microsomes from CD-1 mice required NADPH and was strongly inhibited by carbon monoxide. Lung and liver microsomes isolated from animals treated with phenobarbital demonstrated no changes in covalent binding of [14C]1,1-DCE compared with those from vehicle-treated animals. While 3-methylcholanthrene caused no alterations in binding to lung microsomes, the same pretreatment resulted in significantly increased levels of binding to liver microsomes. Piperonyl butoxide caused significant decreases in covalent binding to lung and liver microsomes; SKF 525-A significantly inhibited binding to liver microsomes but had no effect on lung microsomes. The incubation of liver microsomes with inhibitors required more NADPH than those performed with lung microsomes. The results demonstrate that reactive metabolites of 1,1-DCE can be formed by lung and liver microsomes, and suggest the involvement of cytochrome P-450 isozymes in the lung and liver injury induced by the halocarbon. However, metabolic activation by lung and liver microsomes may additionally involve non P-450 dependent mechanisms as evidenced by relatively high levels of nonspecific binding of 1,1-DCE.

Isolation of an N-alkylprotoporphyrin IX from chick embryo livers following the administration of 3,5-diethoxycarbonyl-1,4-dihydro-4-ethyl-2,6-dimethylpyridine.

The ferrochelatase-lowering activity of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) analogues in chick embryo hepatocyte culture has been assumed to be due to the formation of an N-alkylprotoporphyrin IX. This assumption required confirmation. For this reason the 4-ethyl analogue of DDC was administered to phenobarbital-pretreated 19-day-old chick embryos. This resulted in hepatic accumulation of a green pigment with ferrochelatase-inhibitory activity. The green pigment was identified as an N-alkylprotoporphyrin IX by comparison of the electronic absorption spectra of its dimethyl ester and Zn complex with the corresponding spectra obtained from synthetic N-ethylprotoporphyrin IX.

Effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices.

The effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices have been examined. Methylmercury induced a concentration-dependent increase in the spontaneous release of glutamate, aspartate, gamma-aminobutyric acid, and taurine from mouse cerebellar slices. Glycine release was slightly increased, but not in a concentration-dependent manner. The spontaneous release of glutamine from mouse cerebellar slices was not altered by any concentration of methylmercury examined (10, 20, and 50 microM). The tissue content of glutamate, gamma-aminobutyric acid, glutamine, and taurine decreased after exposure to methylmercury. Exposure of cerebellar slices to 20 microM methylmercury resulted in a significant enhancement in glutamate release during stimulation with 35 mM K+. This increase could be accounted for by the methylmercury-induced increase in spontaneous glutamate release. The increase in spontaneous release of glutamate and gamma-aminobutyric acid was independent of the availability of extracellular calcium. These results suggest that methylmercury increases the release of neurotransmitter amino acids, particularly gamma-aminobutyric acid and glutamate, by acting at intracellular sites to increase release from a neurotransmitter pool. The increase in the potassium-stimulated release of glutamate may reflect an increased sensitivity of the cerebellar granule cell to the effects of methylmercury. It is suggested that alterations in amino acid neurotransmitter function in the cerebellum may contribute to some of the neurological symptoms of methylmercury intoxication.

Furosemide toxicity in isolated mouse hepatocyte suspensions.

Incubation of freshly isolated mouse hepatocytes with 0.5 or 1.0 mM furosemide caused a depletion of cellular acid soluble sulfhydryls to approximately 20-30% of control over the course of 4.5 h. The depletion was accompanied by a reduction in cell viability (indicated by the lactate dehydrogenase latency test) which was significant (P less than 0.05) for 0.5 mM but not for 1.0 mM furosemide at 4.5 h. Ultrastructurally, 0.5 or 1.0 mM furosemide caused cytoplasmic changes including loss of glycogen, disaggregation of polyribosomes, vesiculation of endoplasmic reticulum, and occasional appearance of lamellar bodies consisting of concentric arrays of paired smooth membranes. These concentrations of furosemide also caused cell surface changes, including loss of microvilli, development of an irregular shape compared to the spherical appearance of untreated hepatocytes, and the development of occasional blebs. The appearance of pale staining hydropic cells was indicative of the final stages of cell death. N-Acetylcysteine (6.0 mM) was effective at preventing the depletion of soluble sulfhydryls, the loss of viability, and the ultrastructural effects of 0.5 or 1.0 mM furosemide, suggesting a role for soluble sulfhydryls in the pathogenesis of furosemide hepatotoxicity.

Effects of mercury compounds on the spontaneous and potassium-evoked release of [3H]dopamine from mouse striatal slices.

The effects of mercury compounds on the spontaneous and potassium-evoked release of [3H]dopamine from mouse striatal slices have been examined. All mercury compounds examined produced concentration-dependent increases in the spontaneous release of [3H]dopamine, with an order of potency of methylmercury greater than mercuric (Hg2+) mercury greater than p-choloromercuribenzene sulfonic acid. Methylmercury had no effect on the 25 mM potassium evoked release of [3H]dopamine in the presence of 1.3 mM calcium. However, in calcium-free conditions, methylmercury significantly increased the potassium-evoked release of [3H]dopamine. Mercuric mercury significantly reduced the 25 mM potassium evoked release of [3H]dopamine in the presence of 1.3 mM calcium, and this response was not reversible with brief washing of the tissue. In calcium-free conditions, mercuric mercury significantly elevated the evoked release of [3H]dopamine, similar to the result obtained with methylmercury. It is suggested that mercury compounds alter dopaminergic synaptic function, possibly by disrupting calcium homeostasis or calcium-dependent processes, and that methylmercury and mercuric mercury can have differential effects to alter dopaminergic neurotransmission.

Properties of 17- to 19-day-old chick embryo liver microsomes. Induction of cytochrome P-450, effect of storage at low temperature, and resistance to lipid peroxidation.

Maximal hepatic cytochrome P-450 levels were induced in the 17-day-old chick embryo (four to five times control) with a dose of sodium phenobarbital of 6 mg/egg/day for 2 days. Similar levels of hepatic cytochrome P-450 were induced with a dose of propylisopropylacetamide of 4 mg/egg/day for 1 day. Chick embryo hepatic microsomes from phenobarbital-pretreated or from untreated chick embryos could be stored for periods of 14 days at -70 degrees C without a decrease in cytochrome P-450 levels. Moreover, no significant differences was discerned between the degree of suicidal inactivation of chick embryo hepatic cytochrome P-450 by 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine in fresh and stored microsomes. Unlike rat hepatic microsomes, chick embryo hepatic microsomes do not undergo lipid peroxidative loss of cytochrome P-450 and heme when incubated in the presence of NADPH.