Comparative genotoxicity studies of the flame retardant tris(2,3-dibromopropyl)phosphate and possible metabolites

Tris(2,3-dibromopropyl)phosphate (Tris-BP) was activated to mutagens in the Salmonella/microsome quantitative test system. Liver microsomes from rats pretreated with phenobarbital (PB) increased the mutagenicity of 0.05 mM Tris-BP to 186% of the activity obtained with liver microsomes from untreated rats. The addition of 0.02 mM Tris-BP to V79 Chinese hamster cells co-incubated with liver microsomes from PB-pretreated rats increased the number of mutants by a factor of 9.7. Tris-BP also caused genotoxic and cytotoxic responses in primary monolayers of rat hepatocytes. The relative increase in unscheduled DNA synthesis after treatment with 0.05 mM Tris-BP was 2.3-fold as measured by scintillation counting of radiolabelled thymidine incorporated into DNA of isolated nuclei. The use of hepatocytes isolated from PB-pretreated rats reduced the increases in DNA repair synthesis relatively to that in control cells. Monolayers of hepatocytes from untreated rats co-cultured with Salmonella typhimurium TA100 activated Tris-BP to mutagenic intermediates which were released into the culture medium. The studies with the V79 and liver-cell systems indicate that the reactive intermediates formed from Tris-BP are sufficiently stable and lipophilic to traverse the various membranes from the site of generation to the respective cellular targets. The relative degree of genotoxic responses of bis(2,3-dibromopropyl)phosphate, 2,3-dibromopropylphosphate, tris(2,3-bromopropyl)phosphate, tris(2-bromopropyl)phosphate and 2,3-dibromopropanol in the systems studied did not indicate that these compounds were proximate or ultimate reactive metabolites of Tris-BP in liver-derived activation systems.     

Pharmacokinetics of imipramine are affected by age and sex in rats

Chronic treatment with the antidepressant imipramine (IMI) leads to accumulation of imipramine's major metabolite desmethylimipramine (DMI) in the brain. Juvenile, young and middle-aged female rats, as well as juvenile and young male rats were treated chronically with imipramine (14 days) and analyzed 24 hours later for levels of IMI and DMI in the hypothalamus-preoptic area (HPA) and serum. Older animals of both sexes showed higher levels of DMI than juvenile animals, in both the HPA and serum. Females also had higher DMI levels than males at comparable ages. Analysis of IMI and DMI levels at intervals after a single imipramine injection suggested that the initial metabolism of imipramine is slower in older animals and in females (compared to males). The results indicate that age and gender alter the initial metabolism of imipramine, leading to enhanced accumulation of metabolites during chronic treatment in older animals and in female rats, compared to younger rats and males, respectively.     

Progesterone metabolism by major salivary glands of rat--I. Submandibular and sublingual glands

The metabolism of progesterone by the submandibular and sublingual salivary glands of female (nonpregnant and pregnant) and male rats was studied. The metabolism was in both sexes significantly greater in submandibular than in sublingual glands. Sex differences were not seen in sublingual glands but less metabolism was found in homogenates and microsomal fractions of female (nonpregnant and pregnant) submandibular glands compared to that of males. The metabolism did not differ between pregnant and nonpregnant female rats. The metabolites were mainly 5 alpha-pregnane-compounds. On the basis of the metabolites identified it can be concluded that rat submandibular and sublingual glands contain at least 3 alpha-, 3 beta-, 20 alpha- and 20 beta-hydroxysteroid dehydrogenase, 5 alpha- and 5 beta-steroid hydrogenase and 17 alpha-steroid hydroxylase activity. 5 alpha-steroid hydrogenase activity was significantly higher in all preparations of male submandibular glands than in females. In sublingual glands some enzyme activities showed pregnancy-related decreased.     

Hepatic fructose-metabolizing enzymes and related metabolites: role of dietary copper and gender

The purpose of this study was to further examine the hypothesis that variations in hepatic fructose-metabolizing enzymes between males and females might account for the differences in the severity of copper (Cu) deficiency observed in fructose-fed male rats. Weanling rats of both sexes were fed high-fructose diets either adequate or deficient in copper for 45 days. Cu deficiency decreased sorbitol dehydrogenase activity and dihydroxyacetone phosphate levels and increased glyceraldehyde levels in both sexes. Gender effects were expressed by higher activities of glycerol 3-phosphate dehydrogenase and aldehyde dehydrogenase in male than in female rats and higher levels of dihydroxyacetone phosphate and fructose 1,6-diphosphate (F1,6DP) in female than in male rats. The interactions between dietary Cu and gender were as follows: alcohol dehydrogenase activities were higher in female rats and were further increased by Cu deficiency in both sexes; aldehyde dehydrogenase activities were decreased by Cu deficiency only in male rats; sorbitol levels were higher in male rats and were further increased by Cu deficiency in male rats; fructose 1-phosphate (F1P) levels were increased by Cu deficiency in both sexes, but to a greater extent in male rats; glyceraldehyde 3-phosphate levels were higher in female rats, but were decreased by Cu deficiency in female and increased in male rats. Though most of the examined hepatic fructose-metabolizing enzymes and metabolites showed great differences between rats fed diets either adequate or deficient in Cu, it is the activity of fructokinase and aldolase-B, and the concentrations of their common metabolites, F1P and notably F1,6DP, that could be in part responsible for differences in the severity of pathologies associated with Cu deficiency observed between female and male rats.     

Organ-specific DNA damage of tris(2,3-dibromopropyl)-phosphate and its diester metabolite in the rat.

The organ specificity of tris(2,3-dibromopropyl)phosphate(Tris-BP)-induced DNA damage was investigated in the rat 2 h after a single i.p. injection of 350 mumol/kg. Extensive DNA damage, measured with the alkaline elution method, was found in the kidney, liver and small intestine. Less, but significant DNA damage was detected in the brain, lung, spleen, large intestine and testis. The role of different pathways in the activation of Tris-BP to DNA damaging products was studied in isolated liver and testicular cells. Concentrations as low as 2.5-5 microM Tris-BP caused DNA damage in the hepatocytes, whereas an approximately 10-fold higher concentration was needed in testicular cells to produce a similar amount of DNA damage. Depletion of GSH by diethyl maleate (DEM) did not affect the extent of DNA damage caused by Tris-BP in the liver cells, but blocked the genotoxic effect in testicular cells. Two specifically deuterated Tris-BP analogs, C3D2-Tris-BP and C2D1-Tris-BP, were significantly less potent in causing DNA damage than the protio compound in isolated liver cells and were somewhat less potent in testicular cells. The major urinary metabolite of Tris-BP, bis(2,3-dibromopropyl)phosphate (Bis-BP), was less potent than Tris-BP in causing kidney DNA damage after in vivo exposure. Furthermore, Bis-BP induced substantially less DNA damage in isolated liver and testicular cells. Similar to the effect of DEM on the DNA damage caused by Tris-BP, the DNA damage caused by Bis-BP could be decreased by DEM-pretreatment in testicular cells but not in liver cells. The present study shows that Tris-BP is a potent multiorgan genotoxic agent in vivo. The in vitro data indicate that P-450 mediated metabolism of Tris-BP is more important than activation by glutathione S-transferases of Tris-BP in liver cells, whereas the latter activation pathway seems to be most important in testicular cells.     

Sexual Dimorphism in Development of Kidney Damage in Aging Fischer-344 Rats

BackgroundAging kidneys exhibit slowly developing injury and women are usually protected compared with men, in association with maintained renal nitric oxide.ObjectivesOur purpose was to test 2 hypotheses: (1) that aging intact Fischer-344 (F344) female rats exhibit less glomerular damage than similarly aged males, and (2) that loss of female ovarian hormones would lead to greater structural injury and dysregulation of the nitric oxide synthase (NOS) system in aging F344 rat kidneys.MethodsWe compared renal injury in F344 rats in intact, ovariectomized, and ovariectomized with estrogen replaced young (6 month) and old (24 month) female rats with young and old intact male rats and measured renal protein abundance of NOS isoforms and oxidative stress.ResultsThere was no difference in age-dependent glomerular damage between young or old intact male and female F344 rats, and neither ovariectomy nor estrogen replacement affected renal injury; however, tubulointerstitial injury was greater in old males than in old females. These data suggest that ovarian hormones do not influence these aspects of kidney aging in F344 rats and that the greater tubulointerstitial injury is caused by male sex. Old males had greater kidney cortex NOS3 abundance than females, and NOS1 abundance (alpha and beta isoforms) was increased in old males compared with both young males and old females. NOS abundance was preserved with age in intact females, ovariectomy did not reduce NOS1 or NOS3 protein abundance, and estrogen replacement did not uniformly elevate NOS proteins, suggesting that estrogens are not primary regulators of renal NOS abundance in this strain. Nicotinamide adenine dinucleotide phosphate oxidase-dependent superoxide production and nitrotyrosine immunoreactivity were increased in aging male rat kidneys compared with females, which could compromise renal nitric oxide production and/or bioavailability.ConclusionsThe kidney damage expressed in aging F344 rats is fairly mild and is not related to loss of renal cortex NOS3 or NOS1 alpha.     

Progesterone metabolism by major salivary glands of rat--II. Parotid gland

The metabolism of [4-14C]progesterone in the parotid salivary glands of nonpregnant female, pregnant female and male rats were investigated in vitro. The metabolic activity of the male rats was significantly lower than in either of the female groups. The pregnant group was metabolically more active than the nonpregnant female group, but his differences was not statistically significant. I homogenates and soluble fractions the main metabolite was 20-alpha-hydroxy- 4-pregnen-3-one in female rats. In male rats the main metabolites were 20-alpha-hydroxy-4- pregnen-3-one and 3-alpha-hydroxy-5-alpha-pregnan-20-one in homogenates and 20-alpha-hydroxy-4- pregnen-3-one in soluble fractions. In the microsomal fractions of both sexes polar compounds predominated. The results indicated the presence of at least the following progesterone metabolizing enzymes in art parotid salivary glands; 3-alpha-, 3-beta-, 20-alpha- and 20-beta-hydroxysteroid dehydrogenase, 5-alpha-and 5-beta-steroid hydrogenase and 17-alpha-steroid hydroxylase activities. Ind the homogenates and soluble fractions of female rats 20-alpha-hydroxysteroid dehydrogenase activity was significantly higher than in males.     

Tris-(2,3-Dibromopropyl) Isocyanurate,a New Emerging Pollutant,Impairs Cognition and Provokes Depression-Like Behaviors in Adult Rats

Tris-(2,3-dibromopropyl) isocyanurate (TDBP-TAZTO), an emerging brominated flame retardant, possesses the characteristics of candidate persistent organic pollutants and has displayed toxicity to fish and rodents. TDBP-TAZTO can pass through the blood brain barrier and accumulate in brain. However, the neurotoxicity of TDBP-TAZTO has not yet studied in rodents. We hypothesize that TDBP-TAZTO could induce the neurotoxicity in rat hippocampal neurons. The male adult rats were exposed to TDBP-TAZTO of 5 and 50 mg/kg by gavage, daily for 6 months. TDBP-TAZTO resulted in cognitive impairment and depression-like behaviors, which may be related with TDBP-TAZTO-induced hypothalamic-pituitary-adrenal axis hyperactivation, upregulation of inflammatory and oxidative stress markers, overexpression of pro-apoptotic proteins, downexpression of neurogenesis-related proteins in hippocampus, and hippocampal neurons damage in DG, CA1 and CA3 areas. Our findings suggested that TDBP-TAZTO induces significant hippocampal neurotoxicity, which provokes cognitive impairment and depression-like behaviors in adult rats. Therefore, this research will contribute to evaluate the neurotoxic effects of TDBP-TAZTO in human.     

Influence of gender on tamoxifen-induced biochemical changes in serum of rats

Tamoxifen, the widely prescribed drug in the prevention and therapy of breast cancer, may cause side effects which may be influenced by gender. The present study was undertaken to investigate the impact of gender on tamoxifen-induced toxic and biochemical changes following oral administration of tamoxifen at high dose level of 20 mg/kg once daily for a 2-week period in both male and female rats. The results showed marked increases in serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in female rats. In contrast, treatment with tamoxifen in male animals significantly decreased the activity of ALT, with a tendency for a decrease in serum AST levels. In female rats, a significant reduction in the serum activity of acid phosphatase (ACP) was noted, compared with a non-significant decrease in males. Non-significant changes in serum levels of alkaline phosphatase (ALP) were seen in both sexes. Tamoxifen lowered serum contents of total lipid and total cholesterol in both male and female rats. Serum levels of triglycerides were reduced in female rats as compared to a non-significant decrease in male animals. The serum albumin concentration was decreased in both male and female rats, while total protein was decreased only in female animals. Tamoxifen markedly increased serum levels of creatinine in female rats, compared with a non-significant rise in males. Total serum contents of calcium were similarly reduced in both males and females. This is the first study which points to gender-related differences in tamoxifen-induced toxic and metabolic changes in rats. The results indicated that females are more susceptible than males to tamoxifen toxicity, probably due to the ability of tamoxifen to antagonize the action of estrogen in females.     

Kinetics of 8-2 fluorotelomer alcohol and its metabolites, and liver glutathione status following daily oral dosing for 45 days in male and female rats

Fluorotelomer alcohols (FTOHs) are raw materials used in the manufacture of polymeric and surfactant products. Based on previous findings from single oral dosing in rats with radiolabeled 8-2 FTOH, glutathione (GSH) depletion and/or the presence of perfluorinated/polyfluorinated acids and aldehyde metabolites was hypothesized to account for the hepatocellular lesions observed in male rats from a 90-day subchronic oral dosing study. Further, the reported nephropathy in female rats from the subchronic experiment was hypothesized to have been initiated by a thiol metabolite produced by degradation of GSH conjugates. In the current investigation, the kinetics of 8-2 FTOH and its metabolites along with liver GSH status were evaluated in the rat following daily oral dosing with 8-2 FTOH for 45 days at 5 and 125 mg/kg/day. Liver GSH stores 1-2 h after dosing were unaffected, suggesting that GSH depletion is not likely a relevant mode of action in the liver. The tissue metabolite data indicate that the liver toxicity mode of action is likely associated with elevated levels of perfluoroalkyl acids found in males, since other polyfluorinated metabolites and 8-2 FTOH were present in livers from female rats at comparable or higher levels. Detection of the N-acetyl cysteine conjugate of the unsaturated parent telomer alcohol in urine from female rats and not male rats provides some evidence to support the mechanistic basis for the observed kidney effects. Further, the increasing levels of perfluorooctanoic acid (PFOA) in plasma from female rats over the 45-day dosing phase, while unexpected, may reflect an increased net absorption of 8-2 FTOH, slow elimination of intermediates in the metabolic pathway between 8-2 FTOH and PFOA, or altered kidney clearance. The results of this study have enhanced our understanding of 8-2 FTOH kinetics and metabolism and potential modes of action in the rat, which will guide the design of future studies for FTOHs and our need to define the mechanistic basis for the observed effects.     

Correlation of hepatic cytosolic androgen binding proteins with androgen induction of hepatic microsomal ethylmorphine N-demethylase in the rat

Previous reports have demonstrated the presence of moderate to high affinity binding for androgens in the cytosol of livers from male rats. This binding was significantly lower in female rats or in immature rats of either sex. The hepatic androgen binding protein, which sedimented at approx. 4 S on sucrose density gradients, has been called a receptor which mediates the actions of androgens in the liver. The experiments in the present study were designed to evaluate the hepatic androgen binding protein for characteristics which have been attributed to receptors in other tissues and to correlate the presence of androgen binding with androgen induction of hepatic drug metabolism. In the current studies, we have shown that cytosol from the livers of male rats bound [3H]dihydrotestosterone [( 3H]DHT) and translocated this steroid ligand to the nucleus in a time and temperature dependent manner. Cytosol prelabeled with [3H]DHT, when passed over a column of denatured DNA cellulose, eluted in three radioactive peaks. Two of these peaks were absent when cytosol from livers of female or hypophysectomized males was used. In addition, the presence of high concentrations of hepatic androgen binding correlated well with the ability of androgen to induce ethylmorphine N-demethylase, a marker of microsomal cytochrome P-450-dependent drug metabolism. Values for both parameters were higher in males than in either females or hypophysectomized males. Testosterone treatment induced both parameters in ovariectomized females and 17 beta-estradiol repressed both in males. However, testosterone treatment failed to induce hepatic androgen binding in hypophysectomized males and immature males, both of which are also unresponsive to androgen induction of drug metabolism. The results suggest that one or more hepatic cytosolic androgen binding proteins possess several characteristics associated with steroid receptors in reproductive tract tissue. Furthermore, this binding may be implicated as a mediator for the androgen induction of at least one component of hepatic drug metabolism.     

Tissue distribution,metabolism, and elimination of perfluorooctanoic acid in male and female rats

The elimination, tissue distribution, and metabolism of [1-¹⁴C]perfluorooctanoic acid (PFOA) was examined in male and female rats for 28 days after a single ip dose (9.4 μmol/kg, 4 mg/kg). A sex difference in urinary elimination of PFOA-derived ¹⁴C was observed. Female rats eliminated PFOA-derived radioactivity rapidly in the urine with 91% of the dose being excreted in the first 24 hr. In the same period, male rats eliminated only 6% of the administered ¹⁴C in the urine. The sex-related difference in urinary elimination resulted in the observed difference in the whole-body elimination half-life (t_1/2) of PFOA in males (t_1/2 = 15 days) and females (t_1/2 < 1 day). Analysis of PFOA-derived ¹⁴C in tissues showed that the liver and plasma of male rats and the liver, plasma, and kidney of female rats were the primary tissues of distribution. The relatively high concentration of PFOA in the male liver was further examined using an in situ nonrecirculating liver perfusion technique. It was shown that 11% of the PFOA infused was extracted by the liver in a single pass. The ability of the liver to eliminate PFOA into bile was examined in rats whose renal pedicles were ligated to alleviate sex differences in the urinary excretion of PFOA. In a 6-hr period following IP administration of PFOA, there was no apparent difference in biliary excretion, where both males and females eliminated less than 1% of the PFOA dose via this route. We hypothesized that the sex difference in the persistence of PFOA was due to a more rapid formation of a PFOA-containing lipid (i.e., a PFOA-containing mono-, di-, or triacylglycerol, cholesteryl ester, methyl ester, or phospholipid) in the male rat. Also, the increased urinary elimination of PFOA in females may have been due to increased metabolism to a PFOA-glucuronide or sulfate ester. However, no evidence that PFOA is conjugated to form a persistent hybrid lipid was obtained, nor were polar metabolites of PFOA in urine or bile detected. In addition, daily urinary excretion of fluoride in male and female rats before or after PFOA treatment were similar, suggesting that the parent compound is not defluorinated. Thus, the more rapid elimination of PFOA from female rats is not due to formation of a PFOA metabolite.     

Identification of S-1,2,2-trichlorovinyl-N-acetylcysteine as a urinary metabolite of tetrachloroethylene: bioactivation through glutathione conjugation as a possible explanation of its nephrocarcinogenicity

The elimination and metabolism of [14-C]-tetrachloroethylene (Tetra) was studied in female rats and mice after the oral administration of 800 mg/kg [14-C]-Tetra. Elimination of unchanged Tetra was the main pathway of elimination in both species and amounted to 91.2% of the dose in rats and 85.1% in mice. [14-C]-Carbon dioxide (CO2) was found to be a trace metabolite of [14-C]-Tetra. Only a small part of the applied dose was transformed to urinary (rats = 2.3%, mice = 7.1%) and fecal (rats = 2.0%, mice = 0.5%) metabolites. The urinary metabolites were separated and quantified by high performance liquid chromatography (HPLC) and identified by gas liquid chromatography/mass spectrometry (GC/MS). The following metabolites could be identified: oxalic acid (8.0% of urinary radioactivity in rats, 2.9% in mice), dichloroacetic acid (5.1%, 4.4%), trichloroacetic acid (54.0%, 57.8%), N-trichloroacetyl-aminoethanol (5.4%, 5.7%), trichloroethanol, free and conjugated (8.7%, 8.0%), S-1,2,2-trichlorovinyl-N-acetylcysteine (N-acetyl TCVC) (1.6%, 0.5%), and another conjugate of trichloroacetic acid (1.8%, 1.3%). The structures of the identified metabolites indicate two different pathways operative in Tetra biotransformation: cytochrome P-450-mediated epoxidation forming reactive metabolites in the liver and conjugation of Tetra with glutathione (GSH) catalyzed by glutathione transferase(s). The formation of reactive intermediates by renal processing of the glutathione conjugates may provide a molecular mechanism for the nephrotoxicity and nephrocarcinogenicity of Tetra in male rats.     

Sex dependence of clearance rates of aldosterone and its metabolites from plasma of intact rats.

Following I.V. injection of 3H-aldosterone, the rates of clearance of plasma 3H-radioactivity was demonstrated to be sex-dependent in intact rats. Even though the percentages of CH2Cl2-extractable plasma radioactivity are greater in female than in male rats, the quantities of CH2Cl2-extractable label are similar until 60 min post-injection. However, the quantities of non-extractable, polar metabolites of aldosterone (NEPD) are markedly greater in the plasma of males and rapidly reach peak levels 10 min post-injection of aldosterone. In females, these polar metabolites (NEPD) are rapidly cleared from the blood. After bile-duct cannulation, the rate of excretion of aldosterone radiometabolites was demonstrated to be rapid and sex-dependent. Within 1 hr., female rats excreted via the bile 82% of the injected dose of 3H-aldosterone, compared to 49% in male rats. In both sexes, greater than 95% of the total radioactivity excreted in the bile are non-extractable polar metabolites of aldosterone (NEPD). The sex hormones appear to influence not only the nature of metabolism of aldosterone in the liver, but also the rates of clearance of aldosterone and its metabolites from the plasma into the bile.     

Metabolism of N tau-methylhistidine by mice.

Mice and rats were injected with tracer doses of radioactive N tau-[Me-14C]methylhistidine in order to determine the recovery of the injected radioactivity and the extent of the metabolism of N tau-methylhistidine. In the first 27 h after injection, 96.3, 78.0 and 97.5% of radioactivity was excreted by female mice, male mice and male rats respectively. Recovery after 5 days of collection was 98.4 and 92.8% for female and male mice respectively. However, radioactivity associated with N tau-methylhistidine or its acetylated derivative accounted for 44, 86.5 and 96.0% of the excreted radioactivity for female mice, male mice and rats respectively. In female mice the remaining excreted radioactivity was associated with four major peaks of activity when the metabolites were separated by cation-exchange chromatography. In male mice there were only three of these metabolites present. After chromatographic purification, one metabolite was identified by mass spectroscopy to be 1-methylimidazole-4-acetic acid. Examination of the possible sources of this metabolite indicates that, in mice, N tau-methylhistidine is decarboxylated and enters the chain of reactions common to histamine metabolism. Such extensive metabolism precludes the use of N tau-methylhistidine excretion as an index of myofibrillar protein breakdown in mice.     

The toxic effects in rats of some synthanecine carbamate and phosphate esters analogous to hepatotoxic pyrrolizidine alkaloids

Five synthetic compounds analogous to pyrrolizidine alkaloids have been tested for toxicity in rats. These were the bis-N-ethylcarbamate esters of synthanecines A, B, C and D (Compounds I–IV) and the bis-diethylphosphate ester (V) of synthanecine A. The amino alcohol moiety in each of these had a single 5-membered heterocyclic ring in place of the pyrrolizidine amino alcohol (necine) moiety of natural pyrrolizidine alkaloids.The toxicity of these compounds differed considerably. The synthanecine A carbamate (I) was the most toxic, male and female rats being similarly susceptible. Like many hepatotoxic pyrrolizidine alkaloids, a single dose of compound I caused acute centrilobular necrosis of the liver, chronic hepatotoxicity involving the development of persistent giant hepatocytes, and chronic lung injury. Compound III had similar actions but was less toxic. The synthanecine D carbamate (IV) caused acute liver necrosis but no chronic hepatotoxicity, whereas the synthanecine A phosphate (V) had the opposite effect, with only chronic hepatotoxicity.The different toxic effects were related to the structure and metabolism of the compounds. Doses of compounds I, III and IV associated with a similar degree of acute hepatotoxicity led to similar levels of pyrrolic metabolites in the liver. Compound II, which was not hepatotoxic, gave very little liver pyrrole. The liver level of pyrrolic metabolite from the phosphate ester (V) decreased more rapidly than that from (I), and was not associated with acute toxicity.Antimitotic activity, indicated by the appearance of bizarre giant cells, was shown by compounds capable of forming pyrrolic metabolites which were bifunctional alkylating agents, but not by compound IV, which could only form a monofunctional alkylating agent. Pretreatment with phenobarbitone lowered the susceptibility of rats to compound I and greatly increased the liver level of pyrrolic metabolites associated with acute hepatotoxicity. Some rats given compounds I and III had kidney lesions primarily involving the glomerulus. The results confirm that toxic effects characteristic of many natural pyrrolizidine alkaloids can be reproduced using simplified synthetic analogues, and that such toxicity is associated with pyrrolic metabolites.     

A tubule cell model for ifosfamide nephrotoxicity

Mechanisms leading to ifosfamide (IF)-induced renal damage have not been fully elucidated. Recent work suggests that localized renal tubular metabolism of IF and the production of the nephrotoxic chloroacetaldehyde may lead to nephrotoxicity. Presently no pharmacological method to reduce IF nephrotoxicity has been identified. The objectives of this study were to establish a tubule cell model for IF nephrotoxicity, to verify whether renal proximal tubular cells have the necessary cytochrome P450 (CYP) enzymes to oxidize IF, and whether they can metabolize IF to chloroacetaldehyde. CYP3A, and 2B mRNA and protein were identified in LLCPK-1 cells. The cells metabolized the R- and S-IF enantiomers to their respective 2- and 3-dechloroethylifosfamide metabolites, by-products of chloroacetaldehyde formation. Metabolite production was both time and concentration-dependent. IF did not affect cell viability. In contrast, glutathione-depleted cells showed time and dose-dependent damage. The presence of the relevant CYP enzymes in renal tubular cells along with their ability to metabolize IF to its 2- and 3-dechloroethylifosfamide metabolites suggests that nephrotoxic damage may result from the localized production of chloroacetaldehyde. Glutathione is a major defence mechanism against IF toxicity, thus pharmacological methods for replenishing intracellular glutathione may be effective in modulating IF-induced nephrotoxicity.     

Antipyrine metabolism in the rat by three hepatic monooxygenases

The relative contribution of 3 oxidative reactions of antipyrine to its metabolism in vivo were assessed by comparing male and female rats, and by studying the effects of phenobarbital (PB) and 3-methylcholanthrene (MC). After [N¹⁴CH₃] antipyrine, 11–15% of the dose was excreted as ¹⁴CO₂ in both sexes as a consequence of N-demethylation. PB pretreatment had no effect but MC doubled ¹⁴CO₂ excretion. The other metabolites, 4-hydroxyantipyrine (4OHA) and 3-hydroxymethylantipyrine (3HMA), in urine were determined by thin-layer chromatography. Of the total activity in the urine, 4OHA represented 30% in male and 40% in female rats; 3HMA represented 35% in male and 20% in female rats. The ratio of 4OHA to 3HMA in both sexes increased after PB and MC, the effect being more pronounced with the latter. The results show that the 3 major oxidative pathways of antipyrine metabolism are mediated by different enzymes, almost certainly different forms of cytochrome P450.     

Effect of phosphate supplementation on oxygen delivery at high altitude

In the present communication, effect of low doses of phosphate supplementation on short-term high altitude adaptation has been examined. Studies were carried out in 36 healthy, male, sea-level residents divided in a double blind fashion into drug and placebo treated groups. 3.2 mmol of phosphate were given orally to each subject of the drug treated group once a day for 4 days on arrival at an altitude of 3,500 m. Sequential studies were done in the subjects in both groups on the 3rd, 7th, 14th and 21st day of their altitude stay. Haemoglobin, haematocrit, erythrocyte and reticulocyte counts increased to the similar extent in both groups. Blood pH, pO₂ and adenosine tri-phosphate (ATP) did not differ between the two groups. On 3rd day of the altitude stay, inorganic phosphate and 2,3-diphosphoglycerate (2,3 DPG) levels in the drug treated group increased significantly as compared to the placebo group. No significant difference in inorganic phosphate and 2,3 DPG was observed later on in the two groups. Psychological and clinical tests also indicated that the drug treated subjects felt better as compared to the placebo treated subjects. The present study suggests that low doses of phosphate increases circulating 2,3-DPG concentration which in turn brings about beneficial effect towards short term high altitude adaptation.     

Pharmacokinetics of propetamphos following intravenous administration in the F344 rat

Propetamphos [(E)-l-methylethyl 3[[(ethylamino)methoxyphosphinothioyl]oxy]-2-bu-tenoate], the active ingredient in Safrotin,® is an organophosphate developed by Sandoz, Ltd.® (Switzerland) as an insecticide (1). Although metabolism of propetamphos has been previously investigated (2,3), there is no pharmacokinetic data available in the literature. The current studies were undertaken to investigate the pharmacokinetics of propetamphos following intravenous administration in male and female Fischer 344 (F344) rats. Rats were dosed via an indwelling jugular cannula at a dose of 12 mg/kg (one-tenth the oral LD-50). Blood samples were withdrawn via the cannula at predetermined timepoints to quantitate plasma concentrations of propetamphos over time. Propetamphos is highly bound to plasma proteins (free fraction = 0.06). Free propetamphos concentration in plasma vs. time data were analyzed by noncompartmental methods. The terminal elimination rate constant, λ, was significantly different for males versus females (0.015 min^?1 for males and 0.037 min^?1 for females, p = 0.001). Plasma was cleared of unbound propetamphos at rates of 0.559 ± 0.069 and 0.828 ± 0.181 L/min/kg for males and females (mean ± standard error). Mean residence times (MRTs) for propetamphos in the body for males and females were 28.3 ± 5.7 and 14.4 ± 3.5 min, and the volume of distribution at steady state (Vss) was 14.7 ± 2.6 and 12.3 ± 4.5 L/kg. The differences in these parameters, clearance (CI), MRT, and Vss, were not statistically significant at the p < 0.05 level for males versus females, but MRT was nearly significantly different (p = 0.08). Because of the rapid elimination of propetamphos from plasma following intravenous administration, it is unlikely that propetamphos would bioaccumulate in environmentally exposed animals. Although the pharmacokinetic parameters were not statistically different for males and females in these studies, there was a clear clinical difference in their susceptibility to propetamphos toxicity. Female rats presented with overt signs of organophosphate intoxication, whereas males were only slightly effected. The observed gender-related clinical difference in susceptibility to toxicity suggests that there may be a difference in the extent of elimination due to activation versus detoxication of propetamphos in males and females. Another possible explanation for the clinical difference in propetamphos toxicity is that inhibition of acetyl-cholinesterase by the activated, oxygenated form of propetamphos (propetamphos oxon) may be greater in females than in males.