Modulation of pulmonary p-nitroanisole O-demethylase by intratracheally instilled heavy metal salts

Isolated, ventilated, and perfused rat lung preparations were used to investigate the acute effects of intratracheally applied heavy metal salts on the O-demethylation of p-nitroanisole (NA). The NA O-demethylase activity of the lung was characterized by induction (104%) following pretreatment of rats with β-naphthoflavone (BNF, 80 mg/kg), and by inhibition with SKF-525A (50 μm), metyrapone (0.1 mm), or ventilation of the lungs with CO. Pretreatment of rats with indomethacin (25 mg/kg × 3 days, po) or 3-amino-1,2,4-triazole (1 g/kg, ip) did not alter this activity. The NA O-demethylase was, therefore, suggested to be mixed function oxidase (MFO). Intratracheal instillation of NiCl₂ (0.1 or 1.0 μmol/lung) inhibited the O-demethylase activity (30 or 54%, respectively). The apparent K_m of the reaction (0.138 mm) was doubled by NiCl₂ treatment (0.252 mm), and the V_max was decreased from 26.8 to 20.9 nmol p-nitrophenol/min/g. Cadmium chloride (1.0 μmol/lung) increased the activity by 80%, but the K_m was unchanged. No effect was observed at a dose of 0.1 μmol CdCl₂/lung. At doses of 0.1 or 1.0 μmol/lung, CoCl₂ was without measurable effect on this activity. These data suggest that heavy metals present in the atmosphere can interact with pulmonary MFO in low concentrations to alter the metabolism of xenobiotic compounds.     

Inhibition of mixed-function oxidation in perfused rat liver by fluoroacetate treatment

The effect of fluoroacetate, an inhibitor of the citric acid cycle, on the mixed-function oxidation of p-nitroanisole in isolated perfused livers from fed rats was studied. The citric acid cycle was inhibited by injection of 5 mg/kg sodium fluoroacetate into rats 3 hr prior to liver perfusion experiments. Inhibition of the citric acid cycle was marked by accumulation of citrate (5-fold) and decreases in rates of glycolysis and glycogenolysis by 50–90%. Fluoroacetate treatment inhibited mixed function oxidation in the perfused liver by about 50% without affecting p-nitroanisole O-demethylation by isolated microsomes. Fluorocitrate, at concentrations up to 50 μM, did not inhibit microsomal p-nitroanisole O-demethylation in vitro. These data support the hypothesis that mixed-function oxidation in intact hepatocytes is dependent upon reducing equivalents generated via the citric acid cycle.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in chicken aortas: Monooxygenation,bioactivation to mutagens,and covalent binding to DNA in vitro

The capacity of enzymes present in chicken aortas to catalyze the conversion of polynuclear aromatic hydrocarbons to metabolites that covalently bind to DNA and produce mutations in Salmonella typhimurium auxotrophs is described. Chicken aortic homogenates contained active monooxygenases capable of catalyzing the conversion of benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) to oxidized products as measured by a highly sensitive radiometric assay and high-pressure liquid chromatography. BaP was converted primarily to phenols, while DMBA was converted to hydroxymethyl, phenolic, and diol metabolites. Pretreatment of chickens with Aroclor 1254, 3-methylcholanthrene (3-MC), or 5,6-benzoflavone (5,6-BF) resulted in significant (p < 0.05) increases in the aortic oxidative metabolism of both BaP and DMBA; phenobarbital (Pb) and pregnenolone 16α-carbonitrile (PCN) were ineffective as inducers of chicken aortic monooxygenation. Chicken aortic S-9 homogenates catalyzed the bioactivation of BaP and DMBA to mutagens for S. typhimurium strains TA-98 and TA-1538. Aroclor 1254, 3-MC, 5,6-BF, Pb, or PCN pretreatments did not significantly enhance detectable mutagenesis. When aortic homogenates from untreated chickens were utilized, low specific activities of bound products of BaP, DMBA, or 3-MC to DNA were observed. Pretreatment of chickens with either Aroclor 1254 or 3-MC resulted in 7- to 12-fold increases in specific binding activity. Inhibition of DMBA and BaP metabolism as well as DNA binding was observed when 7,8-benzoflavone (130 μm) or β-estradiol (100 μm) were added to incubation flasks. 1,2-Epoxy-3,3,3-trichloropropane (13 μm) enhanced binding of [³H]BaP and [³H]DMBA to DNA. Additions of glutathione to reaction flasks inhibited ³H binding to DNA, suggesting an important role of aortic nonoxidative metabolism of polycyclic aromatic hydrocarbons.     

Inhibition of cholinesterase activity by isocyanates

Exposure of workers to isocyanates may result in irritation and/or sensitization of the respiratory tract. An immunologic mechanism for sensitization has been presented previously. This investigation explored whether, as a possible mechanism for the irritation reaction, the toxic respiratory effect of isocyanates might be due to their ability to inhibit cholinesterases. Hexamethylene diisocyanate (HDI), hexyl isocyanate (HI), and 2,6-toluene diisocyanate (2,6-TDI) were found to completely inhibit purified human serum cholinesterase when added at molar ratios of 4:1 to 8:1 (isocyanate:enzyme). By contrast, molar ratios of 50:1 or greater were required for 50% enzyme inhibition by 2,4-toluene diisocyanate (2,4-TDI), phenyl isocyanate, or o-tolyl isocyanate. Enzyme inhibition was also achieved by exposure of purified cholinesterase to atmospheres containing 1 ppm isocyanates. Under these conditions, HDI and HI were again the most potent enzyme inhibitors with much less reactivity shown by 2,4-TDI and 2,6-TDI. Under more physiologic conditions, when whole human plasma was the source of cholinesterase, HDI and HI were still potent enzyme inhibitors. However, with the latter two isocyanates, the molar concentrations needed to effect 50% enzyme inhibition suggested affinity labeling by these reagents. The potent cholinesterase inhibition shown by HDI and HI may offer explanation for observed respiratory symptomatology noted upon exposure to these isocyanates.     

Inhibition of intercellular communication by chlordecone (kepone) and mirex in Chinese hamster v79 cells in vitro

Effects of chlordecone (kepone) and mirex on cytotoxicity and intercellular communication were studied in a cell culture system with Chinese hamster V79 cells. Metabolic cooperation between cocultivated 6-thioguanine-sensitive and resistent Chinese hamster V79 cells was used as a measure of intercellular communication. Under the experimental in vitro conditions, kepone was more cytotoxic than mirex. Although both pesticides inhibited intercellular communication between 6-thioguanine-resistant and sensitive cells, their pattern of response was quite different. These data suggest that the reported carcinogenic and teratogenic potentials of these pesticides might be mediated by their action as inhibitors of intercellular communication rather than as mutagens.     

Non-selective decrease of collagen synthesis by cultured fetal lung fibroblasts after non-lethal doses of ethanol

Addition of ethanol to cultured fetal lung fibroblasts resulted in decreases of both collagen and noncollagen protein syntheses. The inhibitory effect of ethanol on protein synthesis was dependent on the concentration of ethanol and the number of treatments with ethanol. Significant inhibition of collagen and noncollagen protein synthesis was observed 3 hr after a single treatment in 0.25% (v/v) ethanol. The maximum inhibitory effect of ethanol on protein synthesis was observed at 6 hr after drug addition. Inhibition of protein synthesis was observed when either proline or glycine was used as the precursor amino acid. An inhibition of alcohol dehydrogenase did not block the ethanol-mediated inhibition of protein synthesis. Ethanol, added to cell cultures throughout the log phase, inhibited cell growth during the late log and stationary phases. Ethanol inhibition of collagen and noncollagen protein synthesis was reversed when the cell cultures were washed and suspended in fresh media for 24 hr. These inhibitory effects of ethanol on macromolecular syntheses were not engendered by killing of cells. The viability of the cells, as indicated by trypan blue exclusion, was not affected significantly at the concentrations of ethanol used. The inhibitory effect of ethanol on protein synthesis also did not originate from drug-mediated inhibition of precursor amino acid uptake. Polysomes isolated from ethanol-treated fibroblasts incorporated proline into protein at a rate which was reduced commensurate with cellular protein synthesis. The resultant inhibition by ethanol of protein synthesis was not attributable to a direct effect of drug on polysomes. Treatment of fetal lung fibroblasts with ethanol also caused a marked inhibition of radioactive thymidine and uridine incorporation, indicating a reduction of both total cellular DNA and RNA synthesis. Accordingly, the decrease of protein synthesis resulted from inhibition of RNA synthesis. Furthermore, messenger RNA synthesis may have decreased since polysomes isolated from ethanol-treated fibroblasts synthesized less protein in the wheat germ cell-free system. Unlike other biochemical variables that were inhibited by ethanol treatment, the level of prolyl hydroxylase activity was elevated significantly. The elevated level of prolyl hydroxylase activity, however, was related neither to the rate of collagen polypeptide synthesis nor to the degree of proline hydroxylation of cellular collagen. The data suggest that the growth-retarding effects of nonlethal doses of ethanol on fetal development may result from inhibition of macromolecular synthesis in fetal fibroblasts.     

Alkylation of DNA in specific hepatic chromatin fractions following exposure to methylnitrosourea or dimethylnitrosamine

This study was designed to assess, qualitatively and quantitatively, the methylation of hepatic DNA isolated from specific chromatin fractions following exposure to carcinogenic alkylating agents, methylnitrosourea (MNU), or dimethylnitrosamine (DMN). Male Sprague-Dawley rats were exposed to 0.15 mmol:kg [methyl-³H]MNU (10 mCi/mmol) or to 0.08 mmol/kg [methyl-¹⁴C]DMN (1.0 mCi/mmol) via gastric intubation. Alkylation of hepatic DNA was assessed at 3, 24, 72, and 168 hr postintubation. Hepatic chromatin was fractionated into portions having characteristics of template-active euchromatin (S2) and template-repressed heterochromatin (P2) by digestion with DNase II followed by MgCl₂ precipitation. At times of peak alkylation following the administration of either carcinogen, the S2/P2 DNA alkylation ratio was greater than 1.0 indicating that alkylation was nonrandom. In addition, the S2/P2 DNA alkylation ratio at peak alkylation following MNU treatment was 1.6 times the DMN ratio, suggesting qualitative differences between DMN- versus MNU-induced alkylation.     

The effect of ionophore A23187 and 2,4-dinitrophenol on the structure and function of cultured liver cells

Adult rat hepatocytes maintained in primary culture have been used as a model system to study cellular injury dependent upon extracellular calcium. Incubation of hepatocytes with ionophore A23187 (1 to 5 μm) resulted in leakage of cytoplasmic enzymes, an increase in the number of cells stained with trypan blue, blebing of the plasma membrane, and changes in mitochondrial structure characterized by mitochondrial swelling. Moreover, a 60% decrease in cellular ATP was observed to precede changes in cellular permeability. These cytotoxic alterations induced by ionophore were dependent upon the presence of extracellular calcium. In contrast, 2,4-dinitrophenol depleted ATP much more extensively and induced extensive swelling of mitochondria at concentrations which failed to induce significant leakage of cytoplasmic enzymes. Cytotoxic changes induced by ionophore were potentiated by 2,4-dinitrophenol but not ethionine which has been shown to cause a reduction in cellular ATP levels. However, ethionine potentiated the cytotoxicity induced by 2,4-dinitrophenol.     

Inhibition of rat testicular heme synthesis and depression of microsomal cytochrome P-450 by estradiol benzoate

Twenty-four hours after a single dose (50 μg, s.c.) of estradiol benzoate (EB), rat testicular microsomal heme and cytochrome P-450 were decreased to 72 and 76% of control levels respectively. Treatment of rats with human chorionic gonadotropin (hCG) resulted in elevated levels of microsomal heme and cytochrome P-450 and increased activity of δ-aminolevulinic acid (ALA) synthase (EC 2.3.1.37). However, the hCG-mediated elevations of testicular microsomal heme and cytochrome P-450 content failed to occur in animals treated with EB. To investigate the possibility that the observed effect of EB was mediated through the pituitary, studies were conducted with hypophysectomized animals. The increased microsomal heme and cytochrome P-450 content mediated by hCG in hypophysectomized animals was again prevented by administration of EB. The elevated activity of testicular mitochondrial ALA synthase produced by hCG in both intact and hypophysectomized animals was not affected by EB. Incorporation of [¹³C]ALA into microsomal heme was depressed 60% 12 hr following a single dose of EB (50 μg, s.c.). These data suggest that EB depresses testicular microsomal heme and cytochrome P-450 content by inhibiting the synthesis of heme at an enzymatic reaction other than ALA synthase.     

Nephrotoxicity of chloroform: metabolism to phosgene by the mouse kidney

In this investigation, we have attempted to determine whether chloroform (CHCl3)-induced nephrotoxicity might be due to its metabolism to phosgene (COCl2) in the kidney. We have found that kidney homogenates from DBA/2J male mice in the presence of glutathione metabolize CHCl3 to 2-oxothiazolidine-4-carboxylic acid (OTZ). This product appears to be formed by the initial trapping of COCl2 by two molecules of GSH to form diglutathionyl dithiocarbonate (GSCOSG). Kidney gamma-glutamyl transpeptidase can rapidly metabolize GSCOSG to N-(2-oxothiazolidine-4-carbonyl)-glycine which is then hydrolyzed, possibly by cysteinyl glycinase to OTZ. The finding that deuterium-labeled chloroform (CDCl3) was less nephrotoxic and depleted less renal GSH than did CHCl3 suggests that the metabolism of CHCl3 to COCl2 may also occur in the kidney in vivo and lead to nephrotoxicity.     

Inhibition of human glutathione S-transferases by 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T)

The phenoxyacid herbicides, 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T), inhibit all known isoenzymes of human liver and erythrocyte glutathione (GSH) S-transferase. However, the maximal inhibition and the I50 values vary significantly for different isoenzymes. GSH peroxidase II activity of GSH S-transferases is also inhibited by both these compounds. These studies suggest that the effect of these compounds on human GSH S-transferases is significantly different from that reported for rat liver enzymes.     

The relationship between misonidazole cytotoxicity and base composition of DNA

The damage induced by electrolytically reduced misonidazole on DNAs of varying base composition has been measured. Damage assessment using a variety of techniques including viscometry, helix renaturation, hydroxyapatite chromatography and agarose gel electrophoresis indicates that damage is related to A + T content, suggesting that misonidazole cytotoxicity involves a specific target in DNA.     

Mutagens from the cooking of food. II. Survey by Ames/Salmonella test of mutagen formation in the major protein-rich foods of the American diet

The formation of mutagens in the major cooked protein-rich foods in the US diet was studied in the Ames Salmonella typhimurium test. The nine protein-rich foods most commonly eaten in the USA—ground beef, beef steak, eggs, pork chops, fried chicken, pot-roasted beef, ham, roast beef and bacon—were examined for their mutagenicity towards S. typhimurium TA1538 after normal ‘household’ cooking (deep frying, griddle/pan frying, baking/roasting, broiling, stewing, braising or boiling at 100–475°C). Well-done fried ground beef, beef steak, ham, pork chops and bacon showed significant mutagen formation. For chicken and beef steak high-temperature broiling produced the most mutagenicity, followed by baking/roasting and frying. Stewing, braising and deep frying produced little mutagen. Eggs and egg products produced mutagens only after cooking at high temperatures (the yolk to a greater extent than the white). Commercially cooked hamburgers showed a wide range of mutagenic activity. We conclude that mutagen formation following cooking of protein-containing foods is a complex function of food type, cooking time and cooking temperature. It seems clear that all the major protein-rich foods if cooked to a well-done state on the griddle (eggs only at temperatures above 225°C) or by broiling will contain mutagens detectable by the Ames/Salmonella assay. This survey is a step towards determining whether any human health hazard results from cooking protein-rich foods. Further testing in both short- and long-term genotoxicity bioassays and carcinogenesis assays are needed before any human risk extrapolations can be made.     

On the capacity of human placental enzymes to catalyze the formation of diols from benzo[a]pyrene

Studies were performed on the oxidative biotransformation of benzo[a]pyrene in fortified preparations of human placental microsomes by analysis with high-pressure liquid chromatography. These investigations revealed that the utilization of substrate concentrations (1–2 × 10^?4m) sufficiently high to assure zero-order reaction kinetics (in terms of the generation of phenolic metabolites) produced a marked inhibitory effect on the formation of dihydrodiols in the same reaction mixtures. Relative quantities of dihydrodiols generated increased with decreasing substrate concentrations between 200 and 2.7 μm. Additions of manganese or ferric ions to reaction mixtures altered the ratios of generated phenols to dihydrodiols but did not provide an explanation for the differences observed in the literature. Identical results were obtained with either ¹⁴C- or ³H-labeled benzo[a]pyrene as substrates. The data suggested the possibility that considerable quantities of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a proximate mutagen/carcinogen, may be generated in vivo by placental tissues of women who smoke.     

Ichthyotoxism by a paralytic toxin produced by marine dinoflagellates of the genus Ceratium: relationship to fraction beta isolated from the sponge Tedania ignis

Two hundred tons of the plankton feeding sardine Cetengraulis edentulus died in March 1982, in Carenero (10 degrees 10' N, 66 degrees 05' W), Venezuela. A fraction was extracted from this fish that was toxic to mice by i.p. injection. The animals died in less than 4 min and showed generalized flaccid paralysis. Gel filtration on Sephadex G15 and Bio Gel P2 showed that the toxicity is related to a fraction that blocks the release of acetylcholine in frog (Rana pipiens) neuromuscular junctions. This toxin is similar in chemical properties and presynaptic effect to fraction beta isolated from the sponge T. ignis by Sevcik and Barboza. Fractions of the same biological action and chemical properties were isolated from plankton samples collected in the area of the ichthyotoxism. The correlation analysis between the presence of toxin and a plankton species in a sample, was carried out with a feasibility index (as %) defined by Sevcik and Mijares. Random samples of plankton (29) were collected in 3 locations (11 degrees 50' N, 68 degrees 15' W; 10 degrees 36' 24' N, 67 degrees 14'7' W and 10 degrees 21' N, 64 degrees 21' W). The correlation carried out over 167 species of phytoplankton present in the samples indicates that the species most likely to be responsible for the production of the fraction beta, in order of feasibility index (in parentheses) are: Ceratium furca (54%), Protoperidinium sp. (1.7%) and Protoperidinium pallidum (1.6%). In some samples a fraction similar to fraction alpha from T. ignis was also found. The identification of the phytoplankton responsible for this fraction is, however, less conclusive. The feasibility indexes are: Protoperidinium sp. (58%), Ceratium inflatum (30%), Podolampas sp. (23%), Ornithocercus steini (21%). The genus Ceratium was the second most abundant in Carenero at the time of the fish death. These results suggest that the toxins isolated from C. edentulus and T. ignis have a planktonic origin.     

Lethal potencies and immunoelectrophoretic profiles of venoms of Vipera bornmulleri and Vipera latifii

Lethality determinations are reported for Vipera bornmulleri and V. latifii. V. latifii venom is significantly more toxic than V. bornmulleri by the i.v. route; by the i.p. and s.c. routes there is little difference between the species. Immunoelectrophoretic profiles indicate close antigenic relationship between venoms of these species and V. palaestinae and Bitis spp.; a more remote relationship with V. russelli and Echis carinatus. Protease activity of V. latifii venom is stronger than that of V. bornmulleri.     

Transplacental induction of mixed-function oxygenases in extra-hepatic tissues by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a dose-dependent induction of a mixed-function oxidase system in fetal and maternal extra-hepatic tissues. At doses of 6 μmg/kg, aryl hydrocarbon hydroxylase (AHH) activity was increased 24-, 22- and 4-fold in fetal lung, kidney and skin, respectively, while maternal lung, kidney and adrenal AHH activity was increased 4-, 2- and 2-fold respectively. High-pressure liquid chromatographic (H.P.L.C.) analysis of benzo(a)pyrene (BP) metabolism after TCDD induction indicated that fetal lung, kidney and skin produced significant quantities of benzo(a)pyrene-7,8-dihydrodiol (BP-7,8-diol), benzo(a)pyrene-4,5-dihydrodiol (BP-4,5-diol) and 9- and 3-phenols of BP. The fetal liver produced benzo(a)pyrene-9,10-dihydrodiol (BP-9,10-diol), BP-4,5-diol, BP-7,8-diol and 9- and 3-phenols of BP. Maternal lung also produced BP-9,10-diol, while maternal adrenal gland yielded primarily the 9-phenol of BP. Epoxide hydratase activity was increased 2- to 3-fold in maternal lung, fetal lung and skin after TCDD pretreatment, but was not affected significantly in liver, kidney or placenta. Treatment of pregnant rats with TCDD increased the covalent binding of BP to DNA in preparations containing maternal liver, lung and placenta as well as fetal liver, lung and skin. Pretreatment with TCDD resulted in increased epoxide hydratase and AHH activities in extra-hepatic tissues but only AHH was increased in hepatic tissues, indicating that the inducing capabilities of TCDD differ from, but share some similarities with, both phenobarbital (PB) and 3-methylcholanthrene (MC). Thus, TCDD appears to provide an exceptionally potent and broad-spectrum transplacental induction of carcinogen-transforming enzymes in extra-hepatic tissues.     

Nephrotoxicity of cis-diamminedichloroplatinum (II) (cis-platinum) and the additive effect of antibiotics: Morphological and functional observation in rats

Basic morphological and functional patterns of cis-platinum nephrotoxicity and the additive effect of combined antibiotics were investigated in male Wistar rats. Nephrotoxicity by cis-platinum is produced in a dose-related fashion and characterized by tubular damage primarily involving the corticomedullary junction. Partial recovery of renal function accompanied by diuresis occurs in 2–3 weeks, however, glomerular filtration rate (GFR) remains at low levels. Histologically, patchy areas of cystic changes remains in the inner cortex, among the areas of relatively normal nephrons. Concomitant use of antibiotics such as aminobenzyl penicillin, sulbenicillin, and cephalothin had no additive effect on platinum nephrotoxicity, from morphological and functional aspects. However, a combination of tobramycin and cis-platinum induced severe renal damage with most animals showing a continuous elevation of blood urea nitrogen and decrease in body weight before death. Histologically, acute tubular necrosis involving both inner and outer cortices was evident. Although cis-platinum is clinically used under enhanced diuresis, GFR and tubular damage have to be carefully monitored throughout the course of cis-platinum treatment. When nephrotoxic antibiotics are being concomitantly prescribed for patients already ingesting cis-platinum, then special precautions should be taken.