Correlations between polychlorinated biphenyl immunotoxicity, the aromatic hydrocarbon locus, and liver microsomal enzyme induction in C57BL/6 and DBA/2 mice

The suppression of the antibody response by polychlorinated biphenyls (PCB) in mice is dependent on the planarity of the PCB molecule and on the expression of the aromatic hydrocarbon (Ah) receptor. In this study, the hypothesis that this form of immunotoxicity is a consequence of the activation of the Ah gene complex and that other compounds which are Ah receptor ligands would also be immunotoxic was tested. 2,2',4,4'-Tetrachlorobiphenyl (TCB), 2,3,3',4,4',5-hexachlorobiphenyl (HCB), phenobarbital (PB), or beta-naphthoflavone (BNF) was given ip to either C57BL/6 (B6,Ahb/Ahb) or DBA/2 (D2, Ahd/Ahd) mice 2 days before immunization with sheep erythrocytes. Organ weights, histopathology, hemagglutinating antibody titers, and the splenic direct antibody plaque-forming cell (PFC) response were evaluated on Day 5. Hepatic aryl hydrocarbon hydroxylase (AHH) induction by these compounds and by 2,2',5,5'-TCB and 3,3',4,4'-TCB was measured as an indicator of Ah receptor binding and subsequent activation of the Ah gene complex by methylcholanthrene-type inducers, while aminopyrine N-demethylase (APND) was measured as an indicator of PB-type induction. 2,2',4,4'-TCB and PB had no effects on the immune parameters of either strain but induced APND activity in both strains. 2,2',5,5'-TCB slightly induced APND activity in B6 mice. 2,3,3',4,4',5-HCB caused a 70% suppression of PFC per spleen, decreased the serum antibody titer, elevated cytochrome P-450 levels (193%), induced both APND (165%) and AHH (217%) activity in B6 mice, but it induced only APND (156%) activity in D2 mice. 3,3',4,4'-TCB elevated cytochrome P-450 levels (210%) and induced both APND (129%) and AHH (321%) activities in B6 mice but only increased APND activities (115%) in D2 mice. BNF elevated cytochrome P-450 (144%), caused a 49% suppression in PFC per spleen, and induced both APND (156%) and AHH (248%) activities but only in B6 mice. These results support the hypothesis that the immunotoxicity caused by halogenated and polycyclic aromatic hydrocarbons is a consequence of activation of the Ah gene complex and suggests that this toxic effect can be initiated by any Ah receptor ligand.     

The effects of organophosphate-induced cholinergic stimulation on the antibody response to sheep erythrocytes in inbred mice

In a previous study, we demonstrated that parathion suppressed both the primary IgM and IgG response to sheep erythrocytes (SRC) in inbred and outbred mice (G. P. Casale, S. D. Cohen, and R. A. DiCapua, 1982, Toxicologist2, 94). Suppression occurred after a dosage which produced cholinergic effects but was absent after a lower dosage which did not produce cholinergic signs. This information suggested that immunosuppression might be mediated indirectly as a result of toxic chemical stress. The present study evaluated the relationship between the anticholinesterase action of parathion, malathion, and dichlorvos (DDVP) and their effects on the primary humoral response to SRC. Male $\text{C57B1}\text{6}$ mice were given a single dose of parathion (16 mg/kg, po), malathion (720 mg/kg, po), or DDVP (120 mg/kg, po) 2 days after immunization with SRC. Two days later, tissues were removed for cholinesterase (CHE) assay and enumeration of splenic antibody-forming cells (PFC). All three compounds produced moderate to severe cholinergic poisoning. DDVP produced cholinergic signs beginning $\text{1}\text{2}$ hr after dosing and lasting $\text{1}\text{2}$ to 1 hr. This profile was associated with a rapid but transient inhibition of brain CHE activity. In contrast, malathion and parathion produced prolonged cholinergic poisoning (4 to 7 hr) and prolonged suppression of brain CHE activity. All three compounds suppressed the primary IgM response. However, when they were given as multiple lower doses, none of the compounds suppressed the primary IgG response. These latter treatments produced no cholinergic signs. The cholinomimetic agent, arecoline (65 mg/kg, ip) produced a short-lived cholinergic crisis but no IgM suppression. Sustained-release arecoline produced prolonged cholinergic poisoning (3 to 5 hr) and reduced the number of IgM PFC to 50% of control. These results demonstrated that organophosphate-induced immunosuppression was associated with severe cholinergic stimulation. The immunosuppression may result from direct action of acetylcholine upon the immune system or it may be secondary to the toxic chemical stress associated with cholinergic poisoning.     

Influence of cysteine upon the glutathione status of isolated rat hepatocytes

Contrary to a previous report [J. Viña, R. Hems and H. A. Krebs, Biochem. J.170, 627 (1978)], we have observed that incubation of isolated rat hepatocytes in a medium containing high concentrations of cysteine did not deplete intracellular glutathione (GSH), but instead caused a net increase of GSH. Hepatocyte-dependent accumulation of the mixed disulfide of cysteine and GSH (CYSSG) in incubation media within a 2-hr period was increased several-fold in medium containing cysteine or cystine compared to methionine-containing medium. Hepatocytes (10⁶ cells/ml) placed in medium containing normal levels of cysteine, cystine, or methionine established about a 1:1 ratio of extracellular GSH to GSSG at a concentration of about 6μM each. These results indicate a possible intracellular-extracellular relationship for glutathione and an intracellular response to the extracellular status of glutathione and cyst(e)ine.     

Enflurane effects on acoustic and photic evoked responses

The effects of incremental doses of the anesthetic agent enflurane, in sensory cortex, mesencephalic reticular formation, inferior colliculus and lateral geniculate body were studied simultaneously on sensory evoked responses in freely behaving rats previously, implanted with 80 μm semimicro-electrodes. Experimentation consisted of EEG recording and averaging of 32 evoked responses before (control), and following 0.5, 1.0, 1.5, 2.5 and 4% enflurane levels. A biphasic dose response pattern of amplitude depression at lower doses which changed to amplitude augmentation at higher doses was observed. The drug level at which the amplitude reversal occurred closely correlated to the initiation of analgesia as determined by sural nerve shock. The reticular formation was most depressed by the lower doses while the sensory cortex was most excited by the higher doses. These effects were best observed with the acoustic evoked responses. The progression of effects with increasing enflurane levels suggests a mechanism of reduced reticular formation control of sensory stimuli coupled with marked cortical neuronal excitability. This study represents the first report of the effects of enflurane on deep, as well as surface CNS structures in the rat, uncomplicated by other anesthetics or paralyzing agents.     

Relationship between cyclic AMP-dependent protein kinase activation and Ca uptake increase of sarcoplasmic reticulum fraction of hog biliary muscles relaxed by cholecystokinin-C-terminal peptides

In hog terminal bile duct cholecystokinin peptides caused an activation of cyclic AMP-dependent protein kinase (A-PK) with cyclic AMP, followed by increase in Ca uptake of sarcoplasmic reticulum fraction (SR-F). By contrast, papaverine showed no activation of A-PK-induced Ca uptake by SR-F with cyclic AMP. The Ca uptake by SR-F was dependent on ATP and Mg², but the component phosphorylated was not the phosphoenzyme intermediate in Ca²-ATPase. The effect of Ca uptake was blocked by the inclusion of a protein inhibitor of A-PK. The correlation coefficient between cyclic AMP-dependent SR-F phosphorylation and stimulated Ca uptake by the phosphorylated SR-F was 0.731 (P < 0.01). These results suggest that one of the mechanisms by which CCK-4, CCK-8, and CCK-33 peptides relax isolated Oddi's sphincters of terminal bile ducts is activation of A-PK-induced Ca uptake by sarcoplasmic reticulum fraction and possibly also by plasma membrane.     

Effects of methionine sulfoximine on the enzymes of glutamate metabolism in isolated astrocytes of rat brain

The enzymes of glutamate metabolism were estimated in astrocytes isolated from brains of normal rats and those injected with the potent convulsant, methionine sulfoximine (MSO), which inhibits glutamine synthetase and induces Alzheimer type II astrocytosis. The wet weight, dry weight; contents of DNA, RNA, protein and the activities of glutamate dehydrogenase and aspartate aminotransferase were elevated following MSO administration. The metabolic effects of MSO were found to be different from those of ammonia wherein a fall in the activity of glutamate dehydrogenase and an increase in the activity of glutamine synthetase was noticed. Based on these results it is suggested that there might be an inverse relationship in the functioning of these two enzymes. Such a relationship would help in preventing the depletion of energy pools in a given cellular compartment during ammonia detoxification.     

The role of glutathione and cytochrome P-450 in the metabolism of methyl chloride

Rat liver microsomes metabolized methyl chloride to formaldehyde at a rate 15-fold less than the rate of benzamphetamine demethylation. The reaction rate was stimulated approximately 2-fold in microsomes from phenobarbital-pretreated rats and was inhibited by addition of SKF-525A, carbon monoxide, metyrapone, and hexobarbital to the microsomal suspension, indicating dependence on cytochrome P-450. The in vivo incorporation of ¹⁴CH₃Cl into liver macromolecules, previously shown to reflect metabolism to CH₃Cl to formate, was not significantly altered by SKF-525A, Aroclor 1254, or 3-methylcholanthrene pretreatment of rats, although pretreatment with phenobarbital produced a 35 and 28% increase in ¹⁴CH₃Cl uptake into liver lipid and acid-insoluble material, respectively. Pretreatment with phenobarbital increased the in vivo metabolism of ¹⁴CH₃Cl to ¹⁴CO₂ (also derived from a formate intermediate) by 19%, but had no effect on urinary metabolites derived from ¹⁴CH₃Cl. SKF-525A inhibited ¹⁴CO₂ production from ¹⁴CH₃Cl by 30% and also had no effect on urinary excretion of ¹⁴C. In contrast, pretreatment with diethylmaleate inhibited ¹⁴CH₃Cl incorporation into liver macromolecules by 70 to 85%, and lowered ¹⁴CO₂ expiration and urinary ¹⁴C excretion by 52 and 60%, respectively. S-Methylcysteine pretreatment produced a similar inhibition of ¹⁴CH₃Cl incorporation and metabolism to ¹⁴CO₂; urinary excretion of ¹⁴C, however, was approximately doubled. ¹⁴CH₃Cl uptake into liver was also stimulated by cysteine pretreatment. These results indicate a strong dependence of CH₃Cl metabolism on tissue nonprotein sulfhydryl content and suggest a possible role for cytochrome P-450 in the in vivo metabolism of CH₃Cl. A scheme for the metabolism of CH₃Cl is postulated which involves initial reaction with glutathione, and sequential metabolism of the conjugate to S-methylcysteine, methanethiol, and formaldehyde.     

The in vivo biotransformation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rat

This study presents evidence for the in vivo biotransformation of TCDD in the rat. Three male rats with indwelling bile loop cannulas were given repeated daily po doses of 15 μg [¹⁴C]TCDD/kg body weight. After either two, four, or six doses, the total output of bile from one rat was collected for 24 hr following the last dose. Biliary ¹⁴C activity was excreted at a rate similar to the excretion of ¹⁴C activity in the feces of normal (noncannulated) rats given po doses of [¹⁴C]TCDD. Therefore it is not likely that enterohepatic recycling plays a significant role in the retention of ¹⁴C activity following a dose of [¹⁴C]TCDD. High-pressure liquid chromatography of the bile from these rats showed the presence of at least eight radioactive peaks and very little, if any, unchanged TCDD. These metabolites were all more polar than TCDD, and the chromatographic profile was altered following incubation of the bile with β-glucuronidase. These data, in conjunction with previous studies, indicate that the metabolic transformation of TCDD in the liver may be the rate-limiting step in the elimination of TCDD from the body.     

Two systems are involved in the sulfobromophthalein uptake by rat liver cells: One is shared with bile salts

It is generally believed that the uptake of sulfobromophthalein by rat liver cells is mediated by a single carrier. Accordingly, kinetic plots obtained using a wide concentration range, failed to give any evidence of heterogeneity in the uptake and gave a K_mof 8.9 ± 2.5 μM and a maximal velocity of 4.2 ± 0.8 nmoles/min/10⁶ cells. Na⁺-taurocholate inhibited competitively, but only partially, sulfobromophthalein uptake by isolated rat liver cells. The taurocholate sensitive component of sulfobromophthalein uptake followed Michaelis Menten kinetics with a K_mof 0.9 μM and a V_max of 0.47 nmole/min/10⁶ cells. Taurocholate inhibition reveals an heterogeneity in the uptake of sulfobromophthalein not shown in classical kinetic plots, indicating that two carriers are involved in the uptake of this dye by rat liver cells. The taurocholate sensitive carrier which displays an affinity for sulfobromophthalein ten times higher than that of taurocholate insensitive one, is probably identical to the Na⁺-independent carrier involved in bile salt uptake.     

The relationship between binding to cytochrome P-450 and metabolism of n-alkyl carbamates in isolated rat hepatocytes

The binding constants of an homologous series of n-alkyl (C₂-C₁₀) carbamates

to the cytochrome P-450 of suspensions of isolated, viable rat hepatocytes have been measured. All the carbamates except ethyl and propyl carbamate produced type I difference spectra and their binding affinities (1/K_s) were found to be directly dependent upon their lipophilicity. These binding affinities were similar to those determined in rat liver microsomes. Maximum development of the binding spectrum in hepatocytes was always within one second of the addition of each carbamate, indicating that for these carbamates membrane permeability was not rate limiting for access to, and metabolism by, cytochrome P-450 and that much of the cells' cytochrome P-450 was unoccupied by endogenous substrates. The major metabolites of C₄-C₈ carbamates were unconjugated ω-1 oxidation products. Below hexyl carbamate only the ω-1 hydroxylated metabolite was observed but for the more lipophilic carbamates the keto metabolite was also a major product. The same products were found in blood after i.p. dosing of rats with hexyl carbamate. A direct relationship was observed between the affinity constant of the carbamate for cytochrome P-450 and the total rate of oxidative metabolism in the ω-1 position. Hydrolysis of the carbamate group was a minor metabolic pathway in contrast to the in vivo situation.     

Differential alterations of cholinergic muscarinic receptors during chronic and acute tolerance to organophosphorus insecticides

Male mice treated for 2 weeks with the anticholinesterase insecticide disulfoton (O,O-diethylS-[2-(ethylthio)-ethyl] phosphorodithioate; 10 mg per kg per day) became tolerant to the hypothermic and antinociceptive effects of disulfoton itself and of oxotremorine, a muscarinic cholinergic agonist. Homogenates of brain and ileum from tolerant animals exhibited reduced binding of the specific muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB). In forebrains of tolerant animals, the number of receptors (B_max) was decreased 40% with no change in the affinity constant. Acetylcholinesterase (AChE) activity was 15% of control. Forty-eight hours after a single injection of disulfoton (10 mg/kg) mice were more resistant than their controls to the hypothermic and antinociceptive effects of a second administration of the same insecticide and of oxotremorine. Tolerance was not present 96 hr after a single administration of disulfoton. A single injection of disulfoton produced 74, 65 and 27% inhibition of AChE activity after 4, 48 and 96 hr respectively. Four hours after a second injection at 48 or 96 hr, 73 or 72% inhibition was found. [³H]QNB binding of animals treated with a single injection of disulfoton and of controls did not differ at either time point. An increase in the K_i for inhibition of [³H]QNB binding by unlabeled oxotremorine was observed in forebrain from mice killed 48 hr after a single injection of disulfoton, indicating a decreased affinity of the muscarinic receptor for agonists. Binding of [³H]oxotremorine-M was decreased significantly 48 hr after a single injection of disulfoton and after chronic treatment. It is suggested that a differential down-regulation of muscarinic receptors occurs in acute and chronic tolerance, involving agonist and antagonist binding sites and depending on duration of exposure.     

Antiarrhythmic effects of disulfiram on epinephrine-induced cardiac arrhythmias in rabbits exposed to trichloroethylene

The effects of disulfiram and its major metabolites, diethyldithiocarbamate and carbon disulfide, on epinephrine-induced cardiac arrhythmias in rabbits exposed to trichloroethylene were examined. Disulfiram (1 g ? 1.35 mmole/kg, po) was given 24 and 6 hr prior to a 1-hr exposure to 6000 ppm trichloroethylene. Disulfiram or its metabolites, diethyldithiocarbamate and carbon disulfide (1.35 mmol/kg, ip), were administered (at similar time intervals) to rabbits exposed for 1 hr to 9000 ppm trichloroethylene. Blood levels of trichloroethylene and its metabolites, trichloroethanol and trichloroacetic acid, were measured at time periods ranging from 7.5 min of exposure to 30 min postexposure. The metabolism of trichloroethylene was inhibited in all treatment groups as indicated by significantly lower blood levels of trichloroethanol and trichloroacetic acid following exposure to 6000 or 9000 ppm trichloroethylene but only rabbits exposed to 6000 ppm trichloroethylene showed significant increases in trichloroethylene blood levels when treated with disulfiram. When challenged with 0.5 to 3.0 μg/kg epinephrine, the disulfiram treatment prevented the epinephrine-induced arrhythmias resulting from exposure to 6000 ppm trichloroethylene. All treatment groups in the first 30 min of exposure to 9000 ppm trichloroethylene exhibited an increased percentage of animals responding with arrhythmias compared to controls. Thirty to sixty minutes of exposure resulted in a significant decrease in the percentage of animals responding with arrhythmias in the disulfiram- and diethyldithiocarbamate-treated groups. Disulfiram, but not diethyldithiocarbamate or carbon disulfide, showed a strong trend toward reducing the percentage of animals responding with arrhythmias after 60 min of exposure to 9000 ppm trichloroethylene. These experiments indicate that disulfiram exhibits a cardioprotective activity not related to its effects on TRI metabolism.     

In vivo acetaldehyde in the brain of the rat treated with ethanol

Ethanol, 4.5 g/kg, was administered intragastrically to rats to determine if acetaldehyde could be detected in brain interstitial fluid. Samples from both blood and brain were collected at half-hour intervals. Brain interstitial fluid samples were collected from both the caudate nucleus and the thalamushypothalamus region using the push-pull perfusion technique. The ethanol and acetaldehyde concentrations in these samples were determined by a head space gas Chromatographie technique. Blood ethanol levels typically ranged from 200 to 400 mg/100ml, while acetaldehyde levels ranged from 15 to 40 μM in blood and 5 to 20 μM in brain fluid. When disulfiram was given to the rats 20 hr prior to ethanol administration, blood acetaldehyde increased to 70–280 μM and brain interstitial fluid acetaldehyde increased to between 25 and 120 μM. Whole brain acetaldehyde levels were also measured after an ethanol dose was given. No acetaldehyde could be detected in whole brain unless the animal had first been treated with disulfiram. These data demonstrate that acetaldehyde does enter the brain, coming into direct contact with the brain cells bathed in the interstitial fluid. The acetaldehyde concentration in the interstitial fluid is higher than that in the brain cells, probably due to its rapid oxidation in the cells catalyzed by aldehyde dehydrogenase.     

Interaction of 4-methylbenzaldehyde with rabbit pulmonary cytochrome P-450 in the intact animal, microsomes, and purified systems. Destructive and protective reactions.

About 50 per cent of rabbit pulmonary cytochrome P-450 is destroyed by treatment of the intact animal, microsomes, or systems reconstituted from purified pulmonary mono-oxygenase components with 4-methylbenzaldehyde. The loss of the cytochrome is accompanied by an equimolar loss of heme. The action of 4-methylbenzaldehyde requires the presence of NADPH and O₂ and appears to result from cytochrome P-450-catalyzed metabolism. Selective destruction of one of the known forms of rabbit pulmonary cytochrome P-450 does not account for the lack of complete destruction of pulmonary P-450 by 4-methylbenzaldehyde; loss of about 50 per cent of each form of the cytochrome occurs in vivo and in reconstituted systems. However, form II is affected to a greater extent than form I when microsomes are incubated with 4-methylbenzaldehyde. The portion of the cytochrome not degraded by 4-methylbenzaldehyde appears to be protected by some factor produced from 4-methylbenzaldehyde during the incubation. This factor also protects against complete destruction of the cytochrome by cumene hydroperoxide     

Reduction in eumelanin by the activation of glutathione reductase and gamma-glutamyl transpeptidase after exposure to a depigmenting chemical

Topical application of 4-tertiary butyl catechol (TBC) causes vitiligo in the skin of man and animals, and previous electron microscopic studies showed pheomelanin formation in the affected areas. In the present study, we investigated changes of enzyme activities, eumelanin content and amount of sulfur in tissue cultured human melanoma cells exposed to the depigmenting chemical. TBC enhanced glutathione reductase activity without changing the eumelanin content by 24 hr after exposure and subsequently (by 42 hr) increased gamma-glutamyl transpeptidase activity and sulfur content in the cells with a decrease in eumelanin content. It is suggested that this chemical alters the types of melanin formed by modulation of these enzyme activities.     

The cardiotoxicity of carbon monoxide as a component of polymer pyrolysis smokes

Groups of adult male rats were repeatedly exposed to the pyrolysis products of a rigid polyurethane foam or of hemlock to which was added 0, 500, or 1000 ppm carbon monoxide under conditions that did not result in depletion of oxygen in the exposure atmosphere. Other groups were exposed to carbon monoxide in air. These exposures were associated with the development of cardiotoxicity which, although mild, was reflected consistently in increased in cardiac-specific plasma creatine phosphokinase activity, in increased numbers of ectopic beats in response to norepinephrine stress, and in the appearance of microscopic focal lesions in the myocardium. In general, the magnitudes of these effects increased as the concentration of carbon monoxide in the exposure atmosphere increased. However, the carbon monoxide content of both mixtures of pyrolysis products, and in particular of the pyrolysis products of the rigid polyurethane foam, was insufficient to account quantitatively for the total cardiotoxicity of these smokes.     

The cardiotoxicity of hydrogen cyanide as a component of polymer pyrolysis smokes

Groups of adult male rats were repeatedly exposed to the pyrolysis products either of a rigid polyurethane foam or of hemlock, to which was added 0 or 200 ppm hydrogen cyanide under conditions that did not result in depletion of oxygen in the exposure atmosphere. Other groups were exposed to hydrogen cyanide in air or in air plus carbon monoxide. Some of the hydrogen cyanide-exposed groups were pretreated with a chlorpromazine/thiosulfate combination protective against cyanide toxicity. Exposure to hydrogen cyanide without protective pretreatment was associated with mild cardiotoxicity as measured by the magnitude of release of cardiac-specific creatine phosphokinase activity and by the number of ectopic heart beats induced by norepinephrine injection. Much of the difference in the cardiotoxicities of polyurethane and hemlock smokes could be related to their different contents of hydrogen cyanide. However, the prevalence of focal histopathologic lesions in the rat hearts did not correlate well with the other two measures of cardiotoxicity, suggesting that more than one mechanism may be responsible for production of these cardiotoxic effects.