Enhanced ratio of linoleic acid to arachidonic acid in erythrocyte phosphatidylcholine in rats during withdrawal from ethanol

Male Sprague-Dawley rats were pair-fed for 8 weeks either an alcohol diet or a control diet made isocaloric with dextrose. Those on the alcohol diet were then fed the control diet for 3 days and both groups were killed. Analysis of the fatty acid composition of the various blood lipids showed that the relative level of 18:2 to 20:4 was significantly greater in the phosphatidylcholine fraction from erythrocytes of rats withdrawn from alcohol as compared to that from control animals. It has been suggested that in alcohol-fed animals the hepatic capacity to produce 20:4 from 18:2 is reduced. Therefore the increase in the ratio of 18:2/20:4 in erythrocyte phosphatidylcholine could serve as an index to detect the liver malfunction and to confirm recent chronic alcohol consumption.     

The metabolism of p-propylanisole in the rat and mouse and its variation with dose

We have studied the metabolism of the synthetic flavouring p-propylanisole in rats and mice and investigated the variation in its metabolism with dose. [methoxy-¹⁴C]-p-Propylanisole was given to female Wistar albino rats orally and male CD-1 mice ip at doses ranging from 0.05–1500 mg/kg body weight (0.2–20 μCi/animal). The urine, faeces and ¹⁴CO₂ in the expired air were collected. The urinary metabolites were separated by solvent extraction, thin-layer chromatography and high-pressure liquid chromatography, and characterized by mass and nuclear magnetic resonance spectroscopy and comparison with authentic samples. Three major ¹⁴C-labelled urinary metabolites were excreted, 1'- and 2'-hydroxy-p-propylanisole and p-methoxyhippuric acid. ¹⁴CO₂ was eliminated in the expired air, arising from oxidative O-demethylation. The relative quantities of the metabolites varied markedly with dose. The percentage of the dose that was O-demethylated fell as the dose increased and the proportion in the form of urinary metabolites increased. The relative proportions of the major urinary metabolites also changed with dose. In view of the great discrepancy between human exposure to p-propylanisole in foods (about 15 μg/day) and the doses used for its toxicological evaluation in animals, these results emphasize the importance of considering dose-dependent metabolism when interpreting the significance for man of animal data obtained at very high doses.     

Identification and mutagenicity of aflatoxicol-M1 produced by metabolism of aflatoxin B1 and aflatoxicol by liver fractions from rainbow trout (Salmo gairdneri) fed beta-naphthoflavone

beta-Naphthoflavone (beta NF) fed to rainbow trout (Salmo gairdneri) at 50 or 500 ppm in the diet, modified the in vitro metabolism of aflatoxin B1 (AFB1) by the postmitochondrial fraction (PMF) of the liver. Production of aflatoxicol (AFL) was significantly less in the 500 ppm beta NF-fed group (33.9 ng/mg protein) than in the control group (45.7 ng/mg protein), aflatoxin M1 production was dependent on the dose of beta NF, being greatest in the 500 ppm beta NF-fed group (48.9 ng/mg protein), intermediate in the 50 ppm beta NF-fed group (3.7 ng/mg protein), and was not detected in controls. A new trout metabolite, 4-hydroxyaflatoxicol (aflatoxicol M1, AFLM1) was also detected in small amounts from in vitro metabolism by liver PMF from beta NF-fed trout. Sufficient quantities of AFLM1 for confirmation of identity by ultraviolet spectra, mass spectra and nuclear magnetic resonance spectra were prepared by biotransformation of AFL using liver microsomes and isolation by HPLC. In a modified Ames mutagen assay with Salmonella typhimurium TA98, ALFM1 was 4.1% as mutagenic as AFB1 in a previous determination. The carcinogenicity of AFLM1 to rainbow trout is expected to be considerably less than that of AFB1.     

Influence of gallic acid esters on drug-metabolizing enzymes of rat liver

The effect of three antioxidants, propyl, octyl and dodecyl gallate, on hepatic drug metabolism in male rats was studied in vivo and in vitro. When fed at a dietary concentration of 1% for 14 days, only dodecyl gallate increased relative liver weight. Cytochrome P-450 content was not influenced, but a slight increase in cytochrome b₅ content was observed after the feeding of propyl gallate. Monooxygenase activity (benzo[a]pyrene-hydroxylase and ethoxycoumarin-deethylase activities) was not affected by propyl or octyl gallate. but a significant decrease in benzo[a]pyrene-hydroxylase activity was apparent in rats fed dodecyl gallate. Study of benzo[a]pyrene-metabolite formation in liver microsome preparations from control and propyl gallate-treated rats showed an overall decrease in metabolite production following gallate treatment, the decrease being statistically significant for the formation of the 9,10-dihydrodiol. Epoxide-hydratase activity was enhanced by a factor of 1·5 in rats fed propyl gallate; glutathione-transferase activity was unaffected. In vitro, the gallates proved to be potent inhibitors of ethoxycoumarin deethylation in liver microsomes from untreated and phenobarbital-treated rats; however, when cytochrome P-448 had been induced by pretreatment with 3-methylcholanthrene, ethoxycoumarin deethylase was less sensitive to the inhibitory action of the gallates.     

Methylation of liver DNA of rat and mouse by N-nitrosodimethylamine formed in vivo from dimethylamine and nitrite

The extent of formation of N-nitrosodimethylamine (NDMA) in the stomachs of rats and mice after simultaneous oral administration of [14C]dimethylamine and potassium nitrite was determined by measuring the methylation of liver DNA. With doses of around 1 mg dimethylamine hydrochloride/kg body weight and 50 mg potassium nitrite/kg body weight, 0.8% of the amine was nitrosated on average. The individual fluctuations ranged from 0.2 to 1.3% in the rat and from 0.2 to 1.9% in the mouse. Simultaneous administration of 50 mg sodium ascorbate (vitamin C)/kg body weight inhibited the nitrosation by about 80% while 50 mg alpha-tocopherol acetate (vitamin E)/kg body weight reduced the nitrosation by about a half. Assuming similar kinetics and conditions of nitrosation in rats and man, a comparison of the formation of NDMA in vivo from dietary dimethylamine and nitrite with the estimated human uptake of preformed NDMA revealed that in vivo formation in the stomach of man is probably negligible.     

Metabolism and distribution of 3,4-epithiobutanenitrile in the rat

The metabolism and distribution of [2-14C]- and 35S-labelled 3,4-epithiobutanenitrile (4ETN), a thiirane occurring naturally in cruciferous vegetables, was studied in the rat. A dose of c. 11 mg 4ETN/kg body weight was rapidly transformed into water-soluble compounds and was mainly excreted in the urine, irrespective of the route of administration (oral or ip). The main metabolite in the urine was identified by gas chromatography-mass spectrometry as a mercapturic acid derivative. Low residual radioactivity demonstrated in organs 72 hr after administration was consistent with an earlier report that the thiirane may function as a weak biological alkylating agent.     

Effect of butylated hydroxytoluene on the disposition of [14C]aflatoxin B1 in the lactating rat

The distribution and metabolism of an ip dose of [14C]aflatoxin B1 (AFB1) were studied in lactating Sprague-Dawley rats fed for the previous 13 days on a diet containing 0.5% butylated hydroxytoluene (BHT). Compared with ingestion of a BHT-free diet, treatment with BHT increased the biotransmission of AFB1 metabolites, predominantly aflatoxin M1 (AFM1), into the mammary gland and its content of milk, decreased AFB1 binding to liver nuclear DNA and enhanced the excretion of water-soluble metabolites of AFB1, all measured 6 hr after an oral dose of [14C]AFB1. These changes are related to the induction by BHT of hepatic enzymes involved in the transformation and detoxification of AFB1. The results suggest that exposure to BHT may protect the lactating animal from the carcinogenic effect of AFB1 but may increase the risk of exposure of the newborn infant to the carcinogenic metabolite AFM1.     

3-hydroxy- and 3-keto-3-phenylpropionic acids: Novel metabolites of benzoic acid in horse urine

The metabolism of benzoic acid has been examined in the horse, using ¹⁴C- and deuterium-labelled compounds. Chromatographic analysis of the urine showed the presence of hippuric acid, benzoyl glucuronide and benzoic acid and a discrete band which accounted for 2% of the dose administered. This material was isolated by solvent extraction and HPLC and, following treatment with diazomethane, examined by GC/MS.The major component of this fraction was 3-hydroxy-3-phenylpropionic acid methyl ester, which was accompanied by very much smaller amounts of cinnamic acid methyl ester and acetophenone. The two latter minor components have been shown to be artefacts produced during workup and analysis. Cinnamic acid methyl ester arises by the thermal decomposition of 3-hydroxy-3-phenylpropionic acid methyl ester on the GC column. It is proposed that acetophenone has formed, during workup, by decarboxylation of 3-keto-3-phenylpropionic acid.It is suggested that 3-hydroxy and 3-keto-3-phenylpropionic acids, which are also endogenous in horse urine, have arisen by an addition of a 2 carbon fragment to benzoyl CoA, in a sequence analogous to the reactions of fatty acid biosynthesis. Some implications of the metabolic interrelationships between xenobiotic acids and fatty acids are discussed.     

Metabolism of anethole. I. Pathways of metabolism in the rat and mouse

The metabolic fate of the naturally occurring food flavouring trans-anethole has been investigated in rats and mice. A single 50-mg/kg dose of trans-[methoxy-14C]anethole was given orally to female Wistar albino rats and by ip injection to male CD-1 mice. The major routes of elimination of 14C were the urine and expired air (as 14CO2). Excretion of 14C in the faeces and as volatile compounds in the expired air was very low (total less than 2% of the dose). Urinary metabolites were separated by solvent extraction, TLC and HPLC and were characterized by MS and GC-MS directly and following methylation or trimethylsilylation, the results being compared where possible with authentic standards. Eleven 14C-containing urinary metabolites were identified in the rat and ten in the mouse. These compounds arose from side-chain oxidation, side-chain cleavage and various conjugations. The major urinary metabolites were two isomers of 1-(4'-methoxyphenyl)propane-1,2-diol, 2-hydroxy-1-methylthio-1-(4'-methoxyphenyl)propane and 4-methoxyhippuric acid, the first three all being excreted as glucuronides. In addition to these 14C-labelled metabolites, 4-hydroxypropenylbenzene, the unlabelled product of oxidative O-demethylation of trans-[14C]anethole, was excreted extensively in urine as the glucuronide.     

The comparative metabolism and toxic potency of aflatoxin B1 and aflatoxin M1 in primary cultures of adult-rat hepatocytes

Both aflatoxin B₁ (AFB₁) and a hydroxylated metabolite, aflatoxin M₁ (AFM₁), were potent cytotoxins and genotoxins to primary cultures of rat hepatocytes. However, AFB₁ stimulated the release of lactate dehydrogenase into the culture medium and the loss of viable cells from the monolayer at lower doses than did AFM₁. The lowest toxic doses of AFB₁ and AFM₁ were 0·05–0·1 and 0·6 μg/ culture, respectively. Genotoxicity, determined by an assay for stimulation of DNA repair, was apparent at lower doses than was cytotoxicity. AFB₁ was again more potent than AFM₁, stimulating DNA repair at 0·025 μg/culture. compared to the lowest genotoxic dose of AFM₁ of 0·05 μg/culture. At higher doses (1·2–2·4 μg/culture) the responses due to both aflatoxins in the cytotoxicity and DNA-repair assays were approximately equal. The metabolism of a low dose (c. 0·17 μg/culture) of [¹⁴C]AFB₁ and [³H]AFM₁ by cultured hepatocytes differed significantly. After 1 hr, 50% of the [¹⁴C]AFB₁ remained unchanged in the culture medium, whereas about 18 hr were required for the same amount of [³H]AFM₁ metabolism to occur. [¹⁴C]AFB₁ was metabolized to AFM₁, to polar metabolites recovered in the aqueous phase after chloroform extraction, and to metabolites covalently bound to hepatocyte macromolecules. [³H]AFM₁ was also metabolized to polar metabolites and to forms bound to macromolecules. The degree of covalent binding of the aflatoxins correlated with their cytotoxicity and genotoxicity at lower doses. After a 24-hr incubation, 12·5% of the dose of [¹⁴C]AFB₁ was covalently bound to macromolecules compared to 1·5% of [³H]AFM₁. Although AFM₁ was less potent than AFB₁ in cytotoxicity, DNA-repair and covalent-binding assays using primary cultures of hepatocytes, AFM₁ was still active at relatively low doses and therefore is probably a potent hepatotoxin in vivo.     

In vitro metabolism of penicillic acid with mouse-liver homogenate fractions

The metaboism of penicillic acid (PA), a carcinogenic mycotoxin, was studied in vitro using subcellular fractions of mouse-liver homogenates. PA reacted with glutathione (GSH) both enzymatically and non-enzymatically. Each reaction was of equal importance. The in vitro metabolism of PA using different hepatic subcellular fractions was essentially non-enzymatic when GSH was absent, but metabolism was strikingly increased when GSH was available. In the microsomal preparation in the presence of GSH 75% of the added PA was biotransformed within 30 min to metabolite(s) that were not extractable with organic solvents. HPLC analysis indicated that the metabolite(s) were more polar than the parent compound.     

Characterization of water-soluble glucuronide and sulphate conjugates of aflatoxin B1. 1. Urinary excretion in monkey, rat and mouse

Water-soluble aflatoxin conjugates prepared from urine samples from rats, mice and rhesus monkeys dosed with [14C]aflatoxin B1 (AFB1) ip or iv were hydrolysed by enzymes (beta-glucuronidase and sulphatase), acid or a combination of both treatments. Different amounts of AFB1 and its metabolites were found in hydrolysates from different sources, indicating the presence of glucuronide, sulphate and possibly mercapturate conjugates of aflatoxins. In addition to aflatoxins M1, P1, Q1 and B2a, AFB1 was frequently identified in the products released from the hydrolysates. These water-soluble aflatoxin conjugates were not mutagenic to Salmonella typhimurium TA98 in the presence of rat-liver S-9 mix. However, chloroform extracts of the hydrolysates from beta-glucuronidase and sulphatase treatment showed mutagenic activity in these bacteria in the presence of S-9 mix. Although very low levels of AFB1 radioactivity were detected in the hydrolysates, the potent mutagenic activity of AFB1 contributed to the high numbers of revertant colonies. AFP1 was detected in urine samples from monkeys that were pretreated with phenobarbital before an iv dose of AFB1. No mutagenic activity was detected in the enzymatic hydrolysate of the sample from these monkeys. The results thus indicate that AFB1 can form glucuronide and/or sulphate conjugate(s) directly and be excreted in the urine.     

Nuclear magnetic resonance‐ and mass spectrometry‐based metabolomics to study maleic acid toxicity from repeated dose exposure in rats

Maleic acid (MA), a chemical intermediate used in many consumer and industrial products, was intentionally adulterated in a variety of starch‐based foods and instigated food safety incidents in Asia. We aim to elucidate possible mechanisms of MA toxicity after repeated exposure by (1) determining the changes of metabolic profile using ¹H nuclear magnetic resonance spectroscopy and multivariate analysis, and (2) investigating the occurrence of oxidative stress using liquid chromatography tandem mass spectrometry by using Sprague–Dawley rat urine samples. Adult male rats were subjected to a 28 day subchronic study (0, 6, 20 and 60 mg kg⁻¹) via oral gavage. Urine was collected twice a day on days 0, 7, 14, 21 and 28; organs underwent histopathological examination. Changes in body weight and relative kidney weights in medium‐ and high‐dose groups were significantly different compared to controls. Morphological alterations were evident in the kidneys and liver. Metabolomic results demonstrated that MA exposure increases the urinary concentrations of 8‐hydroxy‐2′‐deoxyguanosine, 8‐nitroguanine and 8‐iso‐prostaglandin F_2α; levels of acetoacetate, hippurate, alanine and acetate demonstrated time‐ and dose‐dependent variations in the treatment groups. Findings suggest that MA consumption escalates oxidative damage, membrane lipid destruction and disrupt energy metabolism. These aforementioned changes in biomarkers and endogenous metabolites elucidate and assist in characterizing the possible mechanisms by which MA induces nephro‐ and hepatotoxicity.     

Some Preformulation Studies of Pyruvic Acid and Other α-Keto Carboxylic Acids in Aqueous Solution: Pharmaceutical Formulation Implications for These Peroxide Scavengers

The purpose of this study is to assess some of the variables determining the aldol-like condensation of pyruvic acid (1), a peroxide scavenger, in aqueous solution to parapyruvic acid and higher oligomers. Its stability is compared to 3 other α-keto carboxylic acids, 2 with sterically hindered methylene groups alpha to the keto functionality (2-3) and phenylglyoxylic acid (4) with no methylene group. High-performance liquid chromatography, nuclear magnetic resonance, and liquid chromatography mass spectroscopy techniques are used in the kinetics and product analyses. 1 condensation is concentration dependent and base catalyzed above pH 7, consistent with the reaction mechanism proceeding through the attack of the fraction of the methylene group, alpha to the keto group, in its anionic form, at the keto group of a second molecule of 1. The major product is confirmed to be parapyruvic acid, but higher-order oligomers are also observed. All 3 of the other α-keto carboxylic acids 2-4 are considerably less reactive, with 4 being completely stable. Stable solutions of 1 can be prepared by the use of relatively dilute solutions maintained at slightly acidic pH values. 1 prevents the oxidation of methionine on addition of hydrogen peroxide.     

Measurement of glycidol hemoglobin adducts in humans who ingest edible oil containing small amounts of glycidol fatty acid esters

Hemoglobin (Hb) adducts are frequently used to address and/or monitor exposure to reactive chemicals. Glycidol (G), a known animal carcinogen, has been reported to form Hb adducts. Here, we measure G adduct levels in humans who daily ingest DAG oil, an edible oil consisting mainly of diacylglycerol. Since DAG oil contains a small amount of glycidol fatty acid esters (GEs), possible exposure to G released from GEs has been raised as a possible concern. For measurement of Hb adducts, we employed the N-alkyl Edman method reported by Landin et al. (1996) using gas chromatography-tandem mass spectrometry with minor modifications to detect G-Hb adducts as N-(2,3-dihydroxy-propyl)valine (diHOPrVal). Blood samples were collected from 7 DAG oil users and 6 non-users, and then G-Hb adduct levels were measured. G-Hb adducts were detected in all samples. The average level of diHOPrVal was 3.5 ± 1.9 pmol/g globin in the DAG oil users and 7.1 ± 3.1 pmol/g globin in the non-users. We conclude that there is no increased exposure to G in individuals who daily ingest DAG oil.     

Competitive binding to the cytosolic 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor. Effects of structure on the affinities of substituted halogenated biphenyls--a QSAR analysis

The proposed mechanism of action of the toxic halogenated aromatics, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), involves the initial binding to a high-affinity, low-capacity, cytosolic receptor protein. Previous studies have shown that several 4'-halo-2,3,4,5-tetrachlorobiphenyls bind to the TCDD receptor and that a lateral substituent on both phenyl rings is required for activity. Using an extended series of eighteen 4'-substituted-2,3,4,5-tetrachlorobiphenyls as probes, the effects of a variable lateral substituent on receptor binding affinity and the induction of aryl hydrocarbon hydroxylase (AHH) in vivo and in rat hepatoma H-4-II E cells have been determined. For most substituents, there was an excellent correlation between the rank-order potency for receptor binding and the rank-order potency for AHH induction. Based on in vitro binding affinities (EC50 values) of the 4'-substituted tetrachlorobiphenyls, a multiparameter regression equation was formulated correlating the binding constants to physicochemical substituent parameters. For thirteen compounds out of the present series, multiple regression analysis of the binding data led to the following equation: log(1/EC50) = 1.53 sigma + 1.47 pi + 1.09HB + 4.08, r = 0.978. The results suggest that halogen substitution on both phenyl rings is not a requirement for binding and that hydrophobic (pi) and electronic (sigma) substituent constants and a variable for hydrogen bond (HB) formation are significant parameters describing relative binding avidities of this series of substituted biphenyls for the TCDD receptor.     

Disposition,elimination and metabolism of O-ethylO-4-nitrophenyl phenylphosphonothioate after subchronic dermal application in male cats

The metabolism, distribution, and excretion of the insecticide O-ethylO-4-nitrophenyl phenylphosphonothioate (EPN) were studied in the male cat. Each cat was given a daily dermal dose of 0.5 mg/kg [¹⁴C] EPN for 10 consecutive days. Fifteen days after the last dose, the cats had excreted 62% of the cumulative dose in the urine and 10% in the faces. No ¹⁴CO₂ was detected in the expired air. O-Ethyl phenylphosphonic acid (EPPA) was identified as the major urinary and fecal metabolite. Phenylphosphonic acid (PPA) was the second highest metabolite. Only traces of the intact EPN were recovered in the urine and feces. The disposition studies performed 1, 5, 10 and 15 days after the administration of the last dose showed that EPN was the major compound identified in the brain, spinal cord, sciatic nerve, adipose tissue, plasma and kidney. Most of the radioactivity in the liver was identified as EPPA followed by PPA. The time course of plasma EPN, determined after the 10th daily dose was biphasic. The slower process had a half-life of 17.0 days. After tissue distribution was completed, tissue elimination was adequately represented as a single first-order process.     

Absorption of 3-nitropropanol (miserotoxin aglycone) from the compound stomach of cattle

Miserotoxin, the toxic component of certain Astragalus spp. (Leguminosae), was rapidly hydrolyzed to 3-nitropropanol (NPOH) in the rumen of cattle dosed with timber milkvetch (A. miser var. serotinus). The aglycone showed a rapid rate of disappearance from the rumen with an average half-life of 1.24 h. In contrast to the passage of Co-EDTA, which showed an exponential rate of increase in the abomasum, NPOH was not detected in abomasal fluid collected from dosed cattle. Rapid absorption of NPOH from the rumen was shown by plasma levels of 3-nitropropionic acid (NPA) and inorganic nitrite, but conversion of NPOH to NPA was not observed to any significant extent in the rumen.     

Comparison of growth,serum biochemistries and n−6 fatty acid metabolism in rats fed diets supplemented with high-gamma-linolenic acid safflower oil or borage oil for 90 days

Recently, steps have been taken to further developments toward increasing gamma-linolenic acid (GLA) concentration and lowering costs in plant seed oils using transgenic technology. Through identification and expression of a fungal delta-6 desaturase gene in the high linoleic acid safflower plant, the seeds from this genetic transformation produce oil with >40% GLA (high GLA safflower oil (HGSO)). The aim of the study was to compare the effects of feeding HGSO to a generally recognized as safe source of GLA, borage oil, in a 90 day safety study in rats. Weanling male and female Sprague–Dawley rats were fed a semi-synthetic, fat free, pelleted diet (AIN93G) supplemented with a 10% (wt/wt) oil blend containing HGSO or borage oil, with equivalent GLA levels. Results demonstrated that feeding diets containing HGSO or borage oil for 90 days had similar biologic effects with regard to growth characteristics, body composition, behavior, organ weight and histology, and parameters of hematology and serum biochemistries in both sexes. Metabolism of the primary n−6 fatty acids in plasma and organ phospholipids was similar, despite minor changes in females. We conclude that HGSO is biologically equivalent to borage oil and provides a safe alternative source of GLA in the diet.     

The effects of butylated hydroxytoluene on the in vitro metabolism, DNA-binding and mutagenicity of aflatoxin B1 in the rat

Butylated hydroxytoluene pretreatment in the rat enhanced the total in vitro metabolism of aflatoxin B1 by the hepatic postmitochondrial fraction (S-9) and increased the formation of aflatoxin M1, aflatoxin Q1 and a metabolite tentatively identified as the aflatoxin-glutathione conjugate, the latter being the major metabolite produced. Addition of diethyl maleate, a glutathione depletor, to the incubation mix, reduced formation of the conjugate. No significant difference between treated and control animals was observed in the S-9-mediated binding of aflatoxin B1 to calf thymus DNA. However, the mutagenicity of aflatoxin B1 in Salmonella typhimurium TA98 was significantly lower in the presence of S-9 from BHT-treated rats than with S-9 from controls.