Antigenic evaluation of Mycobacterium lepraemurium.

Immunodiffusion analysis of Mycobacterium lepraemurium indicated the presence of at lease six antigens. Comparative analysis of the M. lepraemurium antigen-antibody system with similar systems established for other mycobacterial species, showed that M. lepraemurium shared up to two antigens with other species. Although our observations are in accord with some of the studies on the antigenic mosaic of M. lepraemurium, they are in disagreement with the observations of Stanford (1973) concerning a close serological relationship of this organism to M.avium. This incompatiblity cannot be explained satifactorily at present.

Immunological studies on leprosy: separation and evaluation of the antigens of Mycobacterium leprae.

Chromatographically separated antigens of Mycobacterium leprae were tested for their ability to elicit skin reactions in guinea-pigs sensitised with homologous and heterologous mycobacteria. Of the three antigen-positive fractions obtained, one showed specific activity and the other two cross-reactivity, as indicated by studies of hypersensitivity and passive cutaneous anaphylaxis. The fraction exhibiting specificity contained only one antigen, which was protein in nature, whereas the other two fractions contained more than one antigen and possessed both protein and polysaccharide constituents. Because the single-antigen-containing fraction showed both positive skin and PCA reactivity, the suggestion is made that this fraction may contain either an antigen with two determinants or may contain two antigens that are not easily distinguishable by immunodiffusion methods.

Effect of the fungicide benomyl on xenobiotic metabolism in rats.

The effect of benomyl administered orally (p.o.) and intraperitoneally (i.p.) on the activity of hepatic microsomal mixed-function oxidases (MFOs) was studied in rats. A dose of 100 mg/kg given i.p. reduced the activities of several hepatic drug-metabolizing enzymes 24 h following the treatment. A similar reduction in the activities of the MFOs was also noted 24 h following oral benomyl administration at a dose of 500 mg/kg. Furthermore, in vivo inhibition of drug metabolism by benomyl was demonstrated by increased pentobarbital sleeping-time 24 h after p.o. as well as i.p. dosing. No alterations were found in the serum sorbitol dehydrogenase (SDH) at 24 h after i.p. or oral benomyl indicating a lack of hepatotoxic effect. These results indicate that benomyl shows a route-independent effect on MFOs and is not toxic to the liver.

Toxicological implications of pesticides: their toxic effects on seeds of food plants.

Pesticides are widely used for the protection of economic crops from a variety of noxious pests. The repeated and indiscriminate uses and the extreme stability of certain pesticides have led to their accumulation in plants, animals, soils and sediments, thus effecting widespread contamination of the environment. Soil contaminants are especially serious because they can inhibit or impair the seed germination of our food and feed crops. Seeds can come in close contact with pesticides through processes such as prematurity application, fumigation, seed dressings, and seed treatments. Several reports have indicated the toxic effects of pesticides on seed germination. Possible mechanisms of the toxic action on pesticides during the germination of seeds have been discussed with emphasis on biochemical, histological, and cytological alterations. Bioassay procedures employing seed germination as a smiple, feasible, economical, time-saving indicator of toxicity have been described briefly. Attention is then drawn to the possible potential health hazards arising from the presence of pesticidal chemicals in food plants since the toxicological implications of long term exposure to pesticides are often more far-reaching.

Toxicologic studies of N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide (captan): its metabolism by rat liver drug-metabolizing enzyme system.

The degree of toxicity caused in rats by captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) administered intraperitoneally is greater than that induced by orally administered captan. With regard to its effect on the drug-metabolizing enzymes of rat liver, the activity of aniline hydroxylase and the level of cytochrome P-450 were found to decrease in the treated rats 24 h after a single oral dose (650 mg/kg). The loss was even greater in the animals receiving diethyl maleate 1 h prior to captan. Furthermore, usual increase in the activity of drug biotransformation enzymes seen after phenobarbital treatment appears to decrease in rats dosed with this funaicide. In vitro incubations of rat liver microsomes with captan resulted in a profound loss of cytochrome P-450 and the acitivty of benzphetamine N-demethylase as well as aniline hydroxylase. Although the inhibition of drug-metabolizing enzyme activity by captan was observed in microsomal incubations with or without NADPH, a detectable amount of carbonyl sulfide (COS) was found only in the incubations that contained captan plus NADPH. Carbonyl sulfide appears to arise from a captan-derived metabolite, thiophosgene (CSCl2), which decomposes to COS in aqueous solutions and in the presence of NADPH inhibits the activity of drug biotransformation enzymes.

Toxicological implications of the mixed-function oxidase catalyzed metabolism of carbon disulfide.

The results of these studies have indicated that the decrease in the activity of the hepatic mixed-function oxidase enzyme system and the concentration of cytochrome P-450 seen on incubation of carbon disulfide (CS2) with rat liver microsomes in the presence of NADPH is the result of the binding of the sulfur atom released in the mixed-function oxidase catalyzed metabolism of CS2 to carbonyl sulfide (COS). Moreover, it appears that COS is further metabolized by the mixed-function oxidase enzyme system to CO2 and that, analogous to the metabolism of CS2 to COS, the sulfur atom released in this reaction also binds to the microsomes and inhibits benzphetamine metabolism and decreases the concentration of cytochrome P-450 detectable as its carbon monoxide complex. The results of these studies also suggest that the decrease in the concentration of cytochrome P-450 and the liver damage seen on in vivo administration of CS2 to phenobarbital pretreated rats, is due to the mixed-function oxidase catalyzed release and binding of the sulfur atoms of CS2. The decrease in the concentration of cytochrome P-450 seen on incubation of CS2 with rat liver microsomes in the presence of NADPH does not appear to be the result of destruction of the heme group or its dissociation from the apoenzyme since the total amount of protoheme is unchanged in microsomes which have been incubated with CS2 and NADPH as compared to those not incubated with these compounds.

Alterations in hepatic phase I and phase II biotransformation enzymes by garlic oil in rats.

Studies were conducted to examine the effect of a single and repeated administrations of garlic oil (diallyl sulfide) on Phase I and Phase II biotransformation enzymes in rats. Adult, male Sprague-Dawley rats treated with a single dose of garlic oil (500 mg/kg i.p.) showed a significant depression of hepatic cytochrome P-450, aminopyrine N-demethylase and aniline hydroxylase while microsomal protein content, cytochrome b5, NADPH-cytochrome c reductase, benzphetamine N-demethylase and cytosolic glutathione, S-transferase remained unaffected 24 h following the treatment. Although certain microsomal enzymes were depressed, there was no liver damage caused by garlic oil as judged by the putative serum enzyme test. On the other hand, daily administration of garlic oil (50 mg/kg i.p. for 5 days) produced a significant increase in hepatic cytochrome P-450, aminopyrine N-demethylase and benzphetamine N-demethylase activities, but not in the rest of the aforementioned parameters of biotransformation reactions. These data indicate that the effect of garlic oil on the hepatic drug-metabolizing enzyme system is dose-dependent.

Stimulatory and inhibitory effects of dimethyl sulfoxide on microsomal aniline hydroxylase activity.

Dimethyl sulfoxide (DMSO) at a single dose of 3 ml/kg body wt, administered i.p. to male rats, caused a significant increase in the hepatic microsomal aniline hydroxylase activity. However, the level of cytochrome P-450, the activities of NADPH-cytochrome c reductase, benzphetamine N-demethylase and aminopyrine N-demethylase were unchanged at 24 h post-treatment. DMSO interacted with control rat liver microsomes in vitro and produced a type II spectral change (peak at 420 nm and trough at 392 nm). On the other hand, liver microsomes from DMSO-treated rats gave qualitatively similar spectra, but with a higher magnitude of binding. Liver microsomes from DMSO-treated rats showed a 3.4-fold increase in Vmax for aniline hydroxylase, while Km was found to be the same when compared with control rat liver microsomes. In vitro addition of 6 mM DMSO to microsomal incubations from control and DMSO-treated rats caused a 9-fold and a 25-fold increase in Km, respectively, while Vmax values for aniline hydroxylase were unchanged. When DMSO (6 mM) was incubated with rat liver microsomes in the presence of NADPH, there was formation of formaldehyde. The results suggest an interaction of DMSO with microsomal cytochrome P-450.

Efficacy of activated charcoal and other agents in the reduction of hepatotoxic effects of a single dose of aflatoxin B1 in chickens.

Liver injury caused by a toxic dose of aflatoxin B1 (AFB1) (6 mg/kg, p.o.) in experimental chickens was measured by observing changes in hepatic microsomal cytochrome P-450 and the activity of microsomal benzphetamine N-demethylase and serum glutamic oxaloacetic transaminase (SGOT). However, simultaneous administration of activated charcoal, reduced glutathione (GSH), cysteine, selenium, beta-carotene or fisetin with aflatoxin B1 considerably reduced the toxic injury to liver as measured by the above parameters.

In vivo and in vitro effect of chelating agents on drug metabolizing enzymes of the rat.

Rats given calcium disodium ethylenediaminetetraacetic acid (CaNa2EDTA), diethylenetriaminepentaacetic acid (DTPA), dimercaprol, p-aminosalicylic acid (PAS), or d-penicillamine (penicillamine) i.p. for 7 successive days showed a significant decrease in the activity of hepatic microsomal benzphetamine N-demethylase. There was no appreciable change in the microsomal cytochrome P-450 concentration. In vitro incubation of the chelating drugs with liver microsomes isolated from rats pre-treated with phenobarbital caused no significant loss of the hemoprotein. The decreased rate of benzphetamine metabolism in microsomal preparations from rats, pretreated with the chelating drugs, may be attributed partly to hepatic depletion of essential trace elements by the chelating drugs.

Dose-related inhibition of the drug-metabolizing enzymes of rat liver by the pyrrolizidine alkaloid, monocrotaline.

Adult, male Sprague-Dawley rats were given 0, 10, 20, 40 or 80 mg kg-1 of monocrotaline intraperitoneally and the following toxicity parameters determined 24 h post treatment. Compared with the control none of the doses caused significant change in either the relative liver weight or the hepatic microsomal protein concentration. Microsomal cytochrome P450 content and activities of benzphetamine N-demethylase and aniline hydroxylase did not differ from the control at 10 or 20 mg kg-1 dosage. But, there was a significant loss of cytochrome P450 at 40 and 80 mg kg-1 dosages and decrease in the activity of the two enzymes only at the highest dose. Similarly, the highest dose caused a marked elevation of serum sorbitol dehydrogenase and glutamic pyruvic transaminase activity suggestive of severe liver damage.

Induction of hepatic microsomal drug metabolizing enzyme system by levamisole in male mice.

To examine the effect of levamisole on the hepatic drug metabolizing enzyme system of mice, levamisole at a dose of 20 mg kg-1 day-1 was administered i.p. for 5 days. Compared with the control values, the levamisole treatment significantly increased the amount of cytochrome P450 and cytochrome b5 and the in-vitro activities of aminopyrine N-demethylase, benzphetamine N-demethylase and aniline hydroxylase. In contrast, there was no change in microsomal NADPH-cytochrome c reductase activity in-vitro or in the relative liver weight and microsomal protein content compared with the corresponding values for control mice. Furthermore, in-vivo induction of drug metabolism was demonstrated by decreased pentobarbitone sleeping times after levamisole pretreatment. These results indicate that certain hepatic microsomal mixed function oxidases of mice are induced by levamisole, the drug that is frequently used as an anthelmintic in veterinary medicine and as an immunostimulant drug in human medicine.

Toxic effects of aflatoxin B1 in chickens given feed contaminated with Aspergillus flavus and reduction of the toxicity by activated charcoal and some chemical agents.

Aspergillus flavus NRRL 3357 was grown on enriched long-grain rice for 7-10 days to produce aflatoxin B1 (AFB1). The quantity of AFB1 in moldy rice was determined by thin-layer chromatography using ultraviolet light. When the dried moldy rice powder was fed to day-old Hubbard X Hubbard broiler chicks in unmedicated feed (AFB1 level 10 ppm) for 8 weeks, there was a profound reduction in weight gain and feed consumption. Chickens fed AFB1 developed severe liver damage, as determined by the concentration of hepatic microsomal cytochrome P-450 and by the activities of microsomal benzphetamine N-demethylase and serum glutamic oxalacetic transaminase. However, activated charcoal, reduced glutathione, cysteine, selenium (as sodium selenite), beta-carotene, and fisetin administered orally considerably reduced the toxicity of AFB1 in the experimental chickens.

Toxicity of dietary monensin in quail.

Quail were fed monensin to determine liver damage, as measured by changes in activities of serum enzymes and liver microsomal enzymes. Monensin fed at a therapeutic level of 110 ppm for 2 weeks produced an increase in cytochrome P-450 and cytochrome b5 and induction of the activities of benzphetamine N-demethylase, aminopyrine N-demethylase, and aniline hydroxylase, with no changes in the activities of serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). On the other hand, quail fed 110 ppm, 220 ppm, and 330 ppm monensin in feed for 6 weeks showed a significant rise in SDH and AST activities at 330 ppm but not at 110 ppm and 220 ppm. The manifestations of liver toxicity observed at 330 ppm were accompanied by a significant decrease in all the aforementioned hepatic microsomal mixed-function oxidases. In contrast, quail fed monensin at 110 ppm and 220 ppm for 6 weeks produced no change in these parameters except for benzphetamine N-demethylase, aminopyrine N-demethylase, and aniline hydroxylase, which were significantly increased in birds fed 220 ppm of monensin.

Mechanism of the neurotoxic and hepatotoxic effects of carbon disulfide.

The mechanisms of carbon disulfide toxicity can be divided into two categories; nonmicrosomal and microsomal. The nonmicrosomal pathway involves nonenzymatic spontaneous reaction of carbon disulfide with amino or thiol groups that leads to formation of dithiocarbamates or GSH conjugates as well as inhibition of certain enzymes such as dopamine beta-hydroxylase. These reactions primarily lead to neurotoxic effects. The second mechanism of carbon disulfide toxicity involves its metabolism by hepatic microsomal enzymes to two reactive sulfur atoms that bind covalently to cell macromolecules causing hepatotoxicity. This oxidative metabolism of carbon disulfide has been suggested to be responsible for much of the liver pathology and impairment of liver metabolism of other endogenous substrates as well as exogenous compounds entering the body.

Studies on possible sulfadimethoxine toxicity to liver and liver drug metabolizing enzyme system of goats, quail and rats.

No significant increases in serum SDH, ALT and AST activities were observed in goats and rats receiving oral sulfadimethoxine at 5 times the therapeutic dose. The quail showed significantly higher activities of SDH and ALT when compared to control values. Moderate increases in liver microsomal cytochrome P-450 and aniline hydroxylase activity were observed in goats and quail but no appreciable change in benzphetamine N-demethylase activity was detected in any species. These results suggest a lack of hepatic toxicity of sulfadimethoxine to these species under the reported experimental conditions.

Comparison of the effects of piperine administered intragastrically and intraperitoneally on the liver and liver mixed-function oxidases in rats.

Piperine, a major pungent constituent of black and red peppers, was administered to rats intragastrically and intraperitoneally to study whether it alters the activities of hepatic mixed-function oxidases (MFO) and serum enzymes as specific markers of hepatotoxicity. An intragastric dose of 100 mg/kg of piperine to adult, male Sprague-Dawley rats caused an increase in hepatic microsomal cytochrome P-450 and cytochrome b5, NADPH-cytochrome c reductase, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h following treatment. On the other hand, a 10 mg/kg dose given i.p. exhibited no effect on the activities of the aforementioned parameters of the hepatic drug-metabolizing enzyme system. However, when the intragastric and intraperitoneal doses were increased to 800 mg/kg and 100 mg/kg, respectively, the black pepper alkaloid produced a significant decrease in the levels of cytochrome P-450, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h after treatment. None of the treatments significantly elevated the activities of serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and isocitrate dehydrogenase (ICD), suggesting that piperine is not a hepatotoxic agent.

Experimental induction of chronic aflatoxicosis in chickens by purified aflatoxin B1 and its reversal by activated charcoal, phenobarbital, and reduced glutathione.

Aflatoxin B1 (AFB1) caused dose-dependent reductions in weight gain and feed consumption when day-old Hubbard X Hubbard broiler type chicks were maintained on a diet contaminated with either 0, 2.5, 5, or 10 ppm purified AFB1 for 8 weeks. Although changes in these parameters were detected at the 2.5 and 5 ppm, the most profound changes were evident at 10 ppm contamination. The concentration of cytochrome P-450 in hepatic microsomes, measured at the end of 8 weeks, also showed dose-dependent decreases. Cytochrome P-450 content in chickens receiving 2.5, 5, and 10 ppm AFB1 was 16, 28, and 65%, respectively, less than the control. Microsomal benzphetamine N-demethylase activity was not inhibited by 2.5 or 5 ppm, but ingestion of 10 ppm AFB1 reduced its activity by more than 40%. Serum glutamic oxalacetic transaminase (SGOT) levels of chickens receiving 10 ppm AFB1 increased by more than 100%, indicating substantial liver damage. However, birds simultaneously receiving 10 ppm AFB1 and activated charcoal (.1% in the feed) or either reduced glutathione (.05%) or phenobarbital (.05%, given intermittently) in their drinking water showed a trend of improvement in feed consumption (less than 10% reversal) and weight gain (less than 28% reversal) over the birds receiving 10 ppm AFB1 alone. The results also indicate that the simultaneous presence of these agents with AFB1 considerably prevented the inhibitory effect of AFB1 on the microsomal cytochrome P-450 and benzphetamine N-demethylase activity. Furthermore, these agents were able to provide moderate protection against AFB1-induced liver injury manifested by elevation of SGOT activity.

An overview of aflatoxicosis of poultry: its characteristics, prevention and reduction.

Aflatoxicosis represents one of the serious diseases of poultry, livestock and other animals. The cause of this disease in poultry and other food-producing animals has been attributed to the ingestion of various feeds contaminated with A. flavus. This toxigenic fungus is known to produce a group of extremely toxic metabolites, of which aflatoxin B1 (AFB1) is most potent. Avian species especially chicks, goslings, ducklings and turkey poults are most susceptible to AFB1 toxicity. The toxic effects of AFB1 are mainly localized in liver as manifested by hepatic necrosis, bile duct proliferation, icterus and hemorrhage. Chronic toxicity in those birds is characterized by loss of weight, decline in feed efficiency, drop in egg production and increased susceptibility to infections. The incidence of hepatocellular tumors, particularly in ducklings, is considered to be one of the serious consequences of aflatoxicosis. Even though prevention and avoidance are the best way to control aflatoxicosis, natural contamination of crops with A. flavus is sometimes unavoidable. Such aflatoxin-contaminated feeds can be decontaminated using various methods which mainly focus on physical removal or chemical inactivation of the toxins in the feeds. Moreover, dietary additives such as activated charcoal, phenobarbital, cysteine, glutathione, betacarotene, fisetin and selenium have also been reported to be effective in the reduction of aflatoxicosis in poultry.

Comparative studies on the effect of fenbendazole on the liver and liver microsomal enzymes in goats, quail and rats.

To compare the effect of fenbendazole on the liver and liver microsomal mono-oxygenases of goats, quail and rats, an oral dose of 25 mg/kg was administered to the animals daily for 9 consecutive days. On the tenth day, blood samples and livers were collected from both the control and the treated animals for preparation of serum and microsomes respectively. Determination of the activities of sorbitol dehydrogenase (SDH, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum samples showed that there was no significant increase in the activities of these enzymes in the treated animals as compared to their corresponding controls, suggesting no liver damage. Similarly, no significant difference in the amount of microsomal cytochrome P-450 was found between the control and the treated animals of the same species. Compared to their respective controls, the activities of microsomal benzphetamine N-demethylase and aniline hydroxylase were almost unchanged in the treated goats and rats. However, fenbendazole treatment appeared to enhance the activity of these two microsomal enzymes in quail. The results indicate that fenbendazole is not liver toxic to goats, quail or rats at a dose rate of 25 mg/kg.