Genetic variation in the metabolism of coumarin in mouse liver.

The metabolism of 50 microM [3-14C] coumarin to polar products separated by high performance liquid chromatography (HPLC) and covalently bound metabolites in liver microsomes was compared in a series of inbred strains of mice. Coumarin metabolism to total polar products was higher in female than male mice. In all strains, the coumarin 3,4-epoxidation pathway was the major route of metabolism with o-hydroxyphenylacetaldehyde (o-HPA) as the major metabolite. However, in females, there was a major strain difference in the degree of metabolism to coumarin 7-hydroxylase with DBA/2 and 129 having high 7-hydroxycoumarin formation, CBA/Ca having intermediate levels and the other strains low levels. The differences between the strains was much less pronounced in the male mice. There was also evidence for strain variation in metabolism in the quantities of a number of other coumarin metabolites as detected by HPLC analysis of incubate extracts. However, this variation was of a quantitative nature and relatively small. The metabolism of B6C3F1 hybrid mice, in which coumarin had been identified as carcinogenic in a long-term cancer bioassay, was qualitatively similar to that of the other genotypes. The DBA/2 mouse has been suggested as a model for the metabolism of coumarin in humans. The pattern of metabolism found in this strain is different from most other strains. However, the pattern found for all the mouse strains, including DBA/2, differed appreciably from the profiles for other species including humans in the extent of 7-hydroxylation.

Adult respiratory distress syndrome and gonococcemia.

A case of adult respiratory distress syndrome (ARDS) is described that was secondary to conococcal septicemia. The patient had severe hypoxemia and diffuse pulmonary infiltrates. The diagnosis of disseminated gonorrhea was confirmed by the isolation of Neisseria gonorrhoeae from one of several typical appearing skin lesions. The patient did not require mechanical ventilation and made a prompt recovery after a regimen of antibiotics was started. The association between ARDS and gonococcemia is extremely rare. It occurrence may be related to unusual properties of the gonococcal endotoxin or to the host response.

Comparison of the effects of di-(2-ethylhexyl)adipate on hepatic peroxisome proliferation and cell replication in the rat and mouse.

The effects of di-(2-ethylhexyl)adipate (DEHA) have been compared in female F344 rats and female B6C3F1 mice fed diets containing 0-4.0% DEHA and 0-2.5% DEHA, respectively, for periods of 1, 4 and 13 weeks. In both the rat and mouse treatment with DEHA at all time points produced a dose-dependent increase in relative liver weight and hepatic peroxisome proliferation as demonstrated by the induction of peroxisomal (cyanide-insensitive palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities. The magnitude of induction of peroxisome proliferation was similar in both species. Replicative DNA synthesis was studied by implanting osmotic pumps containing 5-bromo-2'-deoxyuridine during study weeks 0-1, 3-4 and 12-13. After 1 week DEHA treatment hepatocyte labelling index values were increased in rats given 2.5 and 4.0% DEHA and mice given 0.6-2.5% DEHA. While DEHA treatment for 4 and 13 weeks did not increase labelling index values in the rat, a sustained stimulation of replicative DNA synthesis was observed in mice given 1.2 and 2.5% DEHA. The results of this study demonstrate a species difference in the hepatic effects of DEHA, in that at some dose levels DEHA can produce a sustained stimulation of replicative DNA synthesis in mouse but not in rat liver. Sustained cell replication provides a better correlation with the observed formation of liver tumours in chronic studies with DEHA in female mice, but not in female rats, than the magnitude of stimulation of hepatic peroxisome proliferation.

Comparison of the metabolism and disposition of [3-14C]coumarin in the rat and marmoset (Callithrix jacchus).

Male Sprague-Dawley rats and marmosets were given a single oral 25 mg/kg dose of [3-14C]coumarin and the excretion of radioactivity in the expired air, urine and faeces monitored up to 96 h. Excretion profiles were similar in both species with the bulk of the dose being excreted in the urine and faeces within 24 h. Chromatographic analysis of 0-48 h urine samples revealed similar metabolic profiles with only small amounts of unchanged coumarin and very little 7-hydroxycoumarin. Coumarin 7-hydroxylase activity was not detectable in hepatic microsomes from either species. These results demonstrate that the disposition of [3-14C]coumarin was similar in the rat and marmoset, a New World primate, and that both species, unlike man, are poor 7-hydroxylators of coumarin.

Nafenopin, a peroxisome proliferator, depletes hepatic vitamin E content and elevates plasma oxidised glutathione levels in rats.

Male Sprague-Dawley rats were given oral doses of nafenopin (80 mg/kg/d) for up to 28 d. Nafenopin administration resulted in liver enlargement and induction of peroxisomal fatty acid beta-oxidation enzymes (which generate hydrogen peroxide), but little effect was observed on catalase and cytosolic GSH peroxidase was decreased. Hepatic vitamin E levels were depleted to around 50% of control. A small increase in hepatic oxidised glutathione (GSSG) content was paralleled in a time-dependent increase in plasma GSSG. These changes in vitamin E and GSSG levels may represent early indicators of oxidative stress produced in rat hepatocytes by nafenopin and other peroxisome proliferators as a consequence of the increased production of hydrogen peroxide.

Effect of DI-n-pentyl phthalate treatment on testicular steroidogenic enzymes and cytochrome P-450 in the rat.

Treatment of young male rats with dipentyl phthalate (DPP) produced significant decreases in testicular cytochrome P-450, cytochrome P-450 dependent microsomal steroidogenic enzymes (17 alpha-hydroxylase, 17-20 lyase) and in the maximal binding of a natural substrate (progesterone) to testis microsomes. No effect was demonstrated by this compound on hepatic cytochrome P-450 content. Treatment of animals with a phthalate ester not causing testicular atrophy (diethyl phthalate; DEP) produced no significant changes in any of the parameters measured. This effect on the enzymes responsible for androgen production may be important as a mechanism of action involved in the development of phthalate-induced testicular damage.

Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes.

Rat hepatocyte suspensions have been used as a model system for some studies on the mechanism of coumarin-induced hepatotoxicity. Hepatocytes were isolated from male Sprague-Dawley rats and subjected to Percoll centrifugation to obtain preparations with >/=92% viability. Coumarin produced time- and concentration-dependent cytotoxic effects in rat hepatocytes as indicated by loss of cell viability and glutathione depletion. [3-(14)C]Coumarin was metabolized by rat hepatocytes to polar metabolites including o-hydroxyphenylacetic acid and to metabolites that became covalently bound to hepatocyte proteins. The addition of 10 mum-ellipticine significantly reduced coumarin cytotoxicity, coumarin metabolism and covalent binding in rat hepatocytes. These results demonstrate that coumarin-induced liver injury in the rat can be modelled in hepatocyte suspensions and that toxicity appears to be due to one or more cytochrome P-450 generated metabolites.

Toxicity to Chinese hamster ovary (CHO) cells of the products of reaction of butylated hydroxyanisole with nitrite at low pH.

Butylated hydroxyanisole (BHA) was found to react readily with nitrite in acidified physiological saline. With low concentrations of reactants at pH 2, HPLC analysis demonstrated the formation of two products, tert-butylquinone (BQ) and a second, unidentified compound. Neutralized BHA/nitrite reaction mixtures were highly toxic to cultured Chinese hamster ovary (CHO) cells. At non-lethal concentrations, causing some cell cycle delay, there was a statistically significant but variable induction of endoreduplication and tetraploidy and a very weak induction of chromosome breakage. The effects were similar to those of pure BQ. It is suggested that the formation of toxic products from BHA, by an oxidative reaction such as that described with nitrite, might be involved in the mechanism of BHA carcinogenesis in the rat forestomach.

Metabolic disposition of 14C-labelled carmoisine in the rat, mouse and guinea-pig.

The absorption, metabolism and excretion of 14C-labelled carmoisine has been studied in the rat, mouse and guinea-pig. Following administration of a single oral dose of either 0.5 or 50 mg/kg body weight, substantially all of the dose was recovered in the excreta within 72 hr, mainly in the faeces. Although the urinary excretion of radioactivity was similar in the rat and the mouse, the proportion of the radioactivity found in the urine of the guinea-pig was significantly greater than that of the other species at both dose levels. Pretreating male rats with unlabelled colouring in the diet (0.05%, w/w) for 28 days prior to dosing with 14C-labelled colouring had no effect on the route of excretion or the time taken to eliminate the majority of the labelled dose. Following a single oral dose of 14C-labelled colouring to previously untreated rats, mice and guinea-pigs or to rats pretreated as above, no marked accumulation of radioactivity in any tissue was found. Pregnant rats eliminated a single oral dose of 14C-labelled colouring at a similar rate to non-pregnant females, and the concentration of radioactivity in the foetuses was similar to that in the other tissues. Naphthionic acid was the major urinary metabolite in all three species. In the rat and mouse, most of the remaining radioactivity co-chromatographed with 2-amino-1-naphthol-4-sulphonic acid (2-ANS), but in the guinea-pig radioactivity also co-chromatographed with 1,2-naphthoquinone-4-sulphonate (1,2-NQS). Only a trace amount of unchanged carmoisine was detected in the urine of the species examined. Naphthionic acid was also found in the faeces of all three species, but neither carmoisine, 2-ANS or 1,2-NQS was detected. At least five other radioactive metabolites were found in the faecal extracts of all three species, including a substantial amount of a compound with chromatographic properties similar to those of a trace metabolite in the urine. Two of the faecal metabolites were hydrolysed by beta-glucuronidase and sulphatase treatment. In studies on the absorption of carmoisine at concentrations of 50, 500 or 5000 ppm from isolated intestinal loops, no significant absorption was detected in the rat, mouse or guinea-pig.

Protein-related differences in the excretion of nitrosoproline and nitrate by the rat--possible modification of de novo nitrate synthesis.

Male Ola:SD rats were fed purified diets containing 5 or 20% lactalbumin as the source of protein, and the daily urinary excretion of nitrate and nitrosoproline was measured. Animals fed the high-protein diet consistently excreted more nitrate and nitrosoproline than littermates fed the low-protein ration, despite a similar, negligible amount of nitrate in both diets. Furthermore, whereas nitrite administration enhanced nitrosoproline excretion in both diet groups, nitrate administration increased nitrosamine output in the low-protein animals but did not affect nitrosation by rats given the 20% lactalbumin ration. Animals fed the 5% lactalbumin diet produced a smaller volume of urine than did the 20% diet group but other measurements of renal function were comparable for both treatments. The results suggest differences in endogenous nitrosation between rats fed diets high or marginal in protein, possibly reflecting decreased nitrate synthesis in the low-protein group.

Formulation and analysis of food-grade mineral hydrocarbons in toxicology studies.

Methods are presented for the formulation and rapid determination of mineral hydrocarbons (MHCs) in animal diet and tissue. Food grade white oils and low melting point waxes are mixed as liquids with powdered diet. Higher melting point waxes are first powdered using a novel atomization technique before dry mixing with diet. MHCs sufficiently soluble in carbon tetrachloride (CCl4) are determined in diet by ultrasonic solvent extraction, adsorption of polar material on Florisil and analysis of the residue by quantitative Fourier Transform Infra Red (FT-IR) spectroscopy. Quantification in tissue is achieved by aqueous saponification, followed by extraction, clean-up and FT-IR analysis as for diet samples. A 10-fold increase in sensitivity over previous methods is achieved, below 0.002% (w/w) in diet and 0.1 mg/g in tissue. Over 80% of the CCl4 used can be recovered and recycled. Control diet seems to contain approximately 0.003% (w/w) background MHC. The method was modified for one powdered wax, only sparingly soluble in CCl4, high concentrations being extracted from diet by flotation in aqueous cetrimide and determined gravimetrically with a limit of detection of 0.1% (w/w) in diet. Application of these methods to 90-day feeding studies is described, and future developments due to the phasing out of CCl4 are discussed.

Metabolism of [3-14C]coumarin to polar and covalently bound products by hepatic microsomes from the rat, Syrian hamster, gerbil and humans.

The metabolism of 0.19 and 2.0 mM-[3-14C]coumarin to polar products and covalently bound metabolites has been studied with hepatic microsomes from the rat, Syrian hamster, Mongolian gerbil and humans. [3-14C]Coumarin was metabolized by liver microsomes from all species to a number of polar products and to metabolite(s) that became covalently bound to microsomal proteins. The polar products included 3-, 5- and 7-hydroxycoumarins, o-hydroxyphenylacetaldehyde and o-hydroxyphenylacetic acid. Coumarin 7-hydroxylation was observed in all species except the rat. With 0.19 mM-[3-14C]coumarin, 7-hydroxycoumarin was the major metabolite in human liver microsomes, whereas in the other species with 0.19 mM substrate and in all species with 2.0 mM substrate o-hydroxyphenylacetaldehyde was the major metabolite. Of the three animal species studied the gerbil most resembled humans as this species also had a high coumarin 7-hydroxylase activity. The administration of Aroclor 1254 to the rat and Syrian hamster induced both microsomal cytochrome P-450 content and [3-14C]coumarin metabolism. With liver microsomes from all species a good correlation between rates of [3-14C]coumarin metabolism and covalent binding was observed at both substrate concentrations. However, in view of the known species difference between the rat and Syrian hamster in coumarin-induced hepatotoxicity, the present data are not consistent with microsomal coumarin metabolite covalent binding being an indicator of potential liver damage.

Metabolic disposition of 14C-labelled amaranth in the rat, mouse and guinea-pig.

The absorption, metabolism and excretion of orally administered 14C-labelled amaranth has been studied in the rat, mouse and guinea-pig. Following administration of a single oral dose of either 2 or 200 mg/kg, most of the radioactivity was excreted in the urine and faeces in the first 24 hr, and substantially all of the dose was recovered in the excreta within 72 hr. In the rat and mouse, the principal route of excretion was the faeces, whereas in the guinea-pig, urinary excretion accounted for up to 50% of the dose. In the rat and guinea-pig the proportion of the dose excreted in the urine was significantly greater at the lower dose level. No marked accumulation of radioactivity was found in any tissues 72 hr after the administration of the labelled colouring. For all three species most of the radioactivity was shown to be associated with naphthionic acid, with traces of unchanged amaranth and a number of other unidentified metabolites also being detected. In the rat and mouse substantially all of the remaining radioactivity was associated with a single unidentified component. Naphthionic acid was found in the faeces of all three species along with a substantial, but variable, amount of unchanged dye. At least six other radioactive peaks were seen in the chromatograms of faecal extracts; two of these peaks had similar chromatographic properties to the unknown metabolites in the urine, but there was no peak corresponding to 1-amino-2-naphthol-3,6-disulphonic acid (1-ANDSA), previously reported as a urinary metabolite of amaranth. In studies of absorption from isolated loops of small intestine of the rat, mouse and guinea-pig, no significant absorption of amaranth was detected over a 100-fold concentration range (20-2000 ppm).

Effects of Escherichia coli lipopolysaccharide on nitrate synthesis and on nitrosation of proline in rats.

Male Ola:SD rats were fed purified diets containing 5 or 20% lactalbumin as the protein source, with or without concomitant administration of Escherichia coli lipopolysaccharide (50-250 micrograms/kg, ip), and changes in 24-hr urinary nitrate excretion, plasma urea, plasma-nitrate pool size and 24-hr urinary nitrosoproline excretion were measured. Urinary nitrate and urinary 14C-nitrosoproline excretion (after oral [14C]proline administration) were significantly greater for rats receiving the high-protein diet compared with those on the low-protein diet. The co-administration of lipopolysaccharide increased nitrate excretion in both diet groups (although the increase was greatest (relatively) in the animals fed 5% lactalbumin), but did not significantly alter urinary nitrosoproline excretion by either group. Plasma urea concentrations and plasma-nitrate pool size were increased by a high-protein diet and/or lipopolysaccharide administration. These findings suggest that treatments which alter the availability of nitrate in vivo are not necessarily associated with increased nitrosation of proline.

Identification of o-hydroxyphenylacetaldehyde as a major metabolite of coumarin in rat hepatic microsomes.

The metabolism of [3-14C]coumarin has been studied in hepatic microsomes from control (corn-oil treated) and Aroclor 1254-treated (100 mg/kg body weight/day, 5 days, ip) rats. [3-14C]Coumarin metabolites in incubate extracts were separated by HPLC and identified by comparison with the retention times of known coumarin metabolites. The major product produced by incubation of 0.25-2.5 mM-[3-14C]coumarin with both control and Aroclor 1254-induced hepatic microsomes was a novel coumarin metabolite. This novel metabolite was extracted from pooled microsomal incubations, purified by semi-preparative HPLC and identified by mass spectrometry as o-hydroxyphenylacetaldehyde (o-HPA). Some possible pathways for the formation of o-HPA from coumarin are proposed.

Studies on the disposition, metabolism and hepatotoxicity of coumarin in the rat and Syrian hamster.

The hepatotoxicity, metabolism and disposition of coumarin has been compared in male Sprague-Dawley rats and Syrian hamsters. The treatment of rats for 12, 24 and 42 weeks with diets containing 0.2 and 0.5% coumarin resulted in hepatotoxicity and increased relative liver weights. While levels of cytochrome P450 (CYP) and CYP-dependent enzymes were decreased, levels of reduced glutathione (GSH) and activities of UDP glucuronosyltransferase, gamma-glutamyltransferase and GSH S-transferase were increased. In contrast, coumarin produced few hepatic changes in the Syrian hamster. Following a single oral dose of 25 mg/kg [3-14C]coumarin, radioactivity was rapidly excreted by the rat and Syrian hamster with the urine containing 63.5 and 89.9%, respectively, and the faeces 38.0 and 12.4%, respectively, of the administered dose after 96 h. The biliary excretion of radioactivity was greater in the rat than in the Syrian hamster. Analysis of 0-24-h urine samples revealed that both species were poor 7-hydroxylators of coumarin. In the rat, treatment with 0.5% coumarin in the diet for 24 weeks was found to increase the urinary excretion of single oral gavage doses of 25 and 300 mg/kg [3-14C]coumarin. The marked species difference in hepatotoxicity between the rat and Syrian hamster observed in this study may be at least partially attributable to differences in coumarin disposition. However, additional studies are required to elucidate the metabolic pathways of coumarin in both species.

Sex and strain differences in mouse hepatic microsomal coumarin 7-hydroxylase activity.

Hepatic microsomal coumarin 7-hydroxylase activity has been determined in male and female mice of strains A/J, AKR, BALB/c, CBA/Ca, C3H/He, C57BL/6J, DBA/2 and 129. In males, coumarin 7-hydroxylase activity was highest in liver microsomes from DBA/2 mice and lowest in BALB/c mice. With female mice enzyme activity was highest in DBA/2 and 129 strains, intermediate in the CBA/Ca strain and comparatively low in the other five strains. Marked sex differences were observed in coumarin 7-hydroxylase activity with enzyme activity in female animals from strains DBA/2, 129 and CBA/Ca being 4.8-, 6.2- and 4.8-fold higher, respectively, than in male mice. In contrast, only minor sex and strain differences in levels of total microsomal cytochrome P-450 were observed. These results demonstrate marked sex and strain differences in mouse hepatic microsomal coumarin 7-hydroxylase activity. Such differences may be due to variations in particular cytochrome P-450 isoenzymes such as CYP2A5, not all of which can be explained by the known allelic difference in the Cyp2a-5 locus.

Toxicity threshold of quinine hydrochloride following low-level repeated dosing in healthy volunteers.

Following a double-blind, four-way crossover design, 32 healthy volunteers (20 males and 12 females) each consumed lactose placebo, or 80, 120 or 160 mg quinine HCl daily for 21 days. Before dosing and at regular intervals during dosing, blood and urine samples were collected and analysed for quinine HCl. Electrocardiography, heart rate, blood pressure, audiometry, peripheral field, funduscopy, colour vision, visual acuity, electronystagmography (ENG) and test for optokinetic nystagmus were all evaluated before dosing and at selected times during dosing. The results showed that daily consumption of up to 80 mg quinine HCl did not significantly alter physiological, ophthalmic or audiometric responses. ENG determination showed that 12.5% of volunteers given lactose placebo or 80 mg quinine HCl exhibited at least one transitory period of ocular motor oscillations. This phenomenon was observed in 18.8% (P < 0.05) of volunteers with a daily intake of 120 mg quinine HCl or more. However, there was not a significant dose-related correlation between nystagmus and daily intake of quinine HCl. Five volunteers consuming lactose placebo displayed an aberrant ocular flutter that decreased significantly (P < 0.05) as the daily intake of quinine HCl increased. One volunteer showed a change in perception of red/green colour vision after taking 160 mg quinine HCl for 21 days. This study demonstrated that the no-untoward-effect level of quinine HCl is at least 80 mg/day.

A study of the toxic hazard that might be associated with the consumption of green potato tops.

Eating green potatoes has reportedly led to poisoning attributed to potato glycoalkaloids (PGA), primarily alpha-solanine and alpha-chaconine. Concentrations of PGA increase during the greening of potatoes but are reportedly much higher in potato tops (leaves). As it is known that members of the UK Bangladeshi community consume potato tops, a study of the toxic hazard that may be associated with the consumption of green potato tops has been carried out. PGA in seven potato varieties were determined by HPLC. Tubers protected from light contained 0.05-0.65 mg/100 g alpha-solanine and 0.3-0.63 mg/100 g alpha-chaconine. Concentrations in leaf samples ranged from 0.64 to 22.6 mg alpha-solanine/100 g and 0.06 to 55.7 mg alpha-chaconine/100 g. Aqueous leaf extracts were cytotoxic to Chinese hamster ovary cells and lysed human, rat and hamster blood cells with no difference in sensitivity among species. Oral administration of potato tops to rats, mice and Syrian hamsters had no adverse effects at the highest practicable dose. A mixture of alpha-solanine and alpha-chaconine (1:1, w/w) given orally at doses of up to 50 mg/kg body weight to hamsters had no effect, but a single ip injection of 25 mg/kg body weight or greater was lethal, with bleeding in the gut. High concentrations of cytotoxic PGA were found in some potato tops, but their effect in laboratory animals was minimal. It is concluded that the consumption of moderate quantities of potato tops (2-5 g/kg body weight/day) is unlikely to represent an acute health hazard to humans.

Comparison of the metabolism of 7-ethoxycoumarin and coumarin in precision-cut rat liver and lung slices.

The metabolism of 7-ethoxycoumarin and [3-(14)C]coumarin was compared in precision-cut rat liver and lung slices. The lung slices were prepared using an agarose gel instilling technique enabling the production of tissue cylinders followed by lung slices employing a Krumdieck tissue slicer. Both 50 microM 7-ethoxycoumarin and 50 microM [3-(14)C]coumarin were metabolized by rat liver and lung slices. 7-Ethoxycoumarin was converted to 7-hydroxycoumarin (7-HC) which was conjugated with both D-glucuronic acid and sulfate. 7-HC sulfate was the major metabolite formed by both liver and lung slices. [3-(14)C]Coumarin was metabolized by rat liver and lung slices to both polar products and to metabolite(s) that bound covalently to tissue slice proteins. The polar products included unidentified metabolites and 3-hydroxylation pathway products, with only very small quantities of 7-HC being formed. These results demonstrate that precision-cut lung slices are a useful model in vitro system for studying the pulmonary metabolism of xenobiotics. Moreover, the precision-cut tissue slice technique may be employed for comparisons of hepatic and extrahepatic xenobiotic metabolism.