Biomedical subjects

F C Kauffman

Publications and source records attributed to F C Kauffman.

At least 37 records · Page 2Linked to original sources

PubMed1993

The involvement of Kupffer cells in carbon tetrachloride toxicity.

Carbon tetrachloride (CCl4) is a classical pericentral hepatotoxicant; however, precise details of its mechanism of action remain unknown. One possibility is that Kupffer cells participant in this mechanism since CCl4 elevates calcium, and the release of toxic eicosanoids and cytokines by Kupffer cells is calcium-dependent. Therefore, these studies were designed to evaluate the role of Kupffer cells in CCl4 toxicity in the rat in vivo. Kupffer cells were destroyed selectively with gadolinium chloride treatment (10 mg/kg GdCl3 iv) 1 day prior to administration of CCl4 (4 g/kg ig). Twenty-four hours after CCl4 treatment, rats were anesthetized, blood samples were drawn for aspartate aminotransferase (AST) determination, which is indicative of parenchymal cell damage, and trypan blue was infused into the liver to stain the nuclei of dead hepatocytes. AST levels were in the normal range and trypan blue staining was negligible in livers from vehicle- or GdCl3-treated rats. As expected, CCl4 treatment alone elevated AST levels to values over 4000 U/liter and caused massive cell death (60-90 trypan blue-positive cells/pericentral field). In dramatic contrast, the elevation in AST and cell death due to CCl4 were almost completely prevented by GdCl3 treatment. In attempts to understand this phenomenon, metabolic and detoxification pathways were assessed. CCl4 is metabolized via cytochrome P450 II.E.1; however, GdCl3 treatment did not alter this pathway as assessed from p-nitrocatechol formation from the selective substrate, p-nitrophenol. GdCl3 treatment also had no effect on hepatic glutathione levels. On the other hand, GdCl3 treatment significantly reduced infiltration of neutrophils resulting from exposure to CCl4. These data clearly support the hypothesis that Kupffer cells participate in the mechanism of toxicity of CCl4 in vivo, possibly by release of chemoattractants for neutrophils.

Animals↗

PubMed1992

Conjugation of benzo(a)pyrene 7,8-dihydrodiol-9,10-epoxide in infant Swiss-Webster mice.

Benzo(a)pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE), accepted as the ultimate carcinogen of benzo(a)pyrene, has a very short half-life in aqueous solutions yet induces lung tumors when injected into infant mice. To evaluate the possibility that metabolites of BPDE, principally in the form of stable conjugates, contribute to binding to DNA in peripheral tissues, infant mice were injected i.p. with 39 nmol (+/- ) anti-BPDE. One h after injection, 5% of the dose was recovered in serum and appeared mostly as conjugated metabolites (54% as glucuronides and 16% as glutathione conjugates). Amounts of direct acting electrophiles in serum estimated by trapping with DNA comprised less than 0.02% of the injected dose. No more than 10% of the radioactivity in extracts of liver, lung, and kidney was recovered as BPDE. Glutathione conjugates predominated in the liver and lung, whereas glucuronides were the major metabolites in kidney. Radioactivity bound to DNA in liver, lung, and kidney was 21.5, 42.7, and 7.8 pmol/mg, respectively. Despite the rapid conversion of BPDE to stable conjugates, 32P-postlabeling profiles of DNA adducts in lung closely resembled that noted after addition of BPDE directly to lung homogenate. Thus, the reactive intermediate as well as stable conjugates of BPDE may be transported to target tissues where they initiate tumors.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1992

Mutagenicity of benzo(a)pyrenyl-1-sulfate in the Ames test.

Comparison of the mutagenicity of nine isomeric benzo(a)pyrenyl [B(a)P] phenols conjugated with either sulfate or glucuronide was carried out using strain Salmonella typhimurium TA98. Of the nine conjugates tested, only B(a)P-1-sulfate was mutagenic. Accordingly, the mutagenicity of B(a)P-1-sulfate was compared with that of B(a)P and 1-hydroxybenzo(a)pyrene [B(a)P-1-OH] in the presence and absence of rat lung S9 and Aroclor-induced liver S9 with and without an NADPH-generating system. B(a)P-1-sulfate was slightly mutagenic, whereas B(a)P and the 1-hydroxy derivative were nonmutagenic when S9 fractions and NADPH were omitted. Addition of induced liver S9 with NADPH caused mutagenicity with B(a) -1-OH greater than B(a)P greater than B(a)P-1-sulfate. B(a)P-1-sulfate was the only mutagenic species when lung S9 was added. This mutagenicity did not require NADPH. Sodium sulfite, an inhibitor of arylsulfatase, decreased the mutagenicity of B(a)P-1-sulfate. These data suggest that a unique mutagenic species is generated from B(a)P-1-sulfate via arylsulfatase in rat lung.

Animals↗

PubMed1992

Involvement of nonparenchymal cells in oxygen-dependent hepatic injury by allyl alcohol.

Allyl alcohol injury to hepatocytes in the perfused liver is oxygen-dependent. It is not known if this injury involves direct action of allyl alcohol on hepatocytes or requires participation of other cell types (e.g., Kupffer cells) present in the liver. Accordingly, the action of allyl alcohol (100-500 microM) on isolated hepatocytes was studied using cells maintained at either 95 or 21% O2. Allyl alcohol toxicity, as indexed by trypan blue uptake, lactate dehydrogenase release, and ATP content, did not differ in the two groups of cells, suggesting that O2 dependency of allyl alcohol toxicity involves other cell types. Administration of allyl alcohol (30 or 40 mg/kg, ip) to rats caused extensive hepatic necrosis localized primarily to periportal regions. To test the involvement of Kupffer cells in the genesis of this injury, male rats (200-350 g) were treated with gadolinium chloride (GdCl3, 10 mg/kg, iv) which diminishes Kupffer cell function and number. The extent of hepatic damage assessed by light microscopy and serum enzymes, aspartate aminotransferase and alanine aminotransferase, was markedly attenuated by pretreatment of rats with GdCl3 24 hr prior to allyl alcohol injection. Thus, O2-dependent hepatic necrosis caused by allyl alcohol involves the presence of Kupffer cells. Since GdCl3 did not prevent toxicity in the perfused liver, circulating blood elements may also contribute to injury of the liver by allyl alcohol in vivo.

1-Propanol↗

PubMed1992

Reversible and irreversible oxidant injury to PC12 cells by hydrogen peroxide.

A simple and reproducible model to identify biochemical changes associated with the transition from reversible to irreversible oxidant injury and cell death was established using rat pheochromocytoma PC12 cells. Cells were subjected to a transient oxidative stress induced by exposure to hydrogen peroxide (H2O2). Reversible loss of high-energy phosphates, induced by exposing cells to 0.2 mM H2O2, was preceded by transient increases in cytosolic calcium with no loss of plasma membrane integrity, as indexed by release of cytosolic enzymes. In contrast, permanent loss of high-energy phosphates, induced by treating cells with 0.5 mH H2O2, was associated with sustained rises in cytosolic-free calcium and increased oxidation of pyruvate and palmitate, two mitochondrial substrates. Initial production of pyruvate and lactate was inhibited by exposure to 0.5 mM H2O2 but returned to values comparable to control values at one hour after treatment with H2O2. Compromise of the plasma membrane was a late event, occurring between 1 and 2 hours after exposure to 0.5 mM H2O2. Collectively, these data indicate that irreversible loss of high-energy phosphates and cell death caused by oxidative stress is more closely associated with altered mitochondrial function than with impaired glycolysis.

Adenosine Triphosphate↗

PubMed1992

Food restriction increases detoxification of polycyclic aromatic hydrocarbons in the rat.

It is well known that food restriction diminishes tumor formation, but mechanisms responsible are difficult to define because multiple physiological changes result from dietary alterations. Studies in this report were designed to focus specifically on the effects of food restriction on hepatic metabolism of polycyclic aromatic hydrocarbons following liver transplantation. By placing livers from food-restricted and untreated rats into naive controls, the effects of diet could be restricted to the liver. After a 15 min infusion of [3H]benzo[a]pyrene under these conditions, food restriction increased polar metabolites in liver (70 pmol/g) and blood (8 pmol/ml) compared to controls approximately 2-fold. Four hours after liver transplantation, levels of polar metabolites in blood were diminished by approximately 50% but were still approximately 2-fold higher in the food-restricted than in the control group. Lung, kidney, spleen, adrenal, ovary, colon, heart and brain also contained higher levels of polar metabolites in the food-restricted than in the control group. The more hydrophobic glucuronides and sulfate conjugates accounted for most of the elevation in polar metabolites in blood from the food-restricted group. In spite of the increase in circulating metabolites in blood of food-restricted animals, DNA binding in liver, lung and kidney was identical in tissues from control and food-restricted groups. In order to evaluate the hypothesis that food restriction stimulated the release of hepatic benzo[a]pyrene metabolites, a liver perfusion model was employed. Maximal rates of release of polar metabolites into the effluent perfusate were approximately 30 and approximately 45 nmol/g/h in livers of control and food-restricted rats respectively. Moreover, rates of metabolism of the model compound p-nitroanisole and glucuronidation of p-nitrophenol were also approximately 2-fold higher in livers from food-restricted than control rats. However, rates of monooxygenation were the same in microsomes prepared from livers of food-restricted or control animals. These results support the hypothesis that food restriction enhances the supply of cofactors which stimulate metabolism of polycyclic aromatic hydrocarbons. This detoxification process may be an important mechanism involved in the protective action of reduced food intake.

Animals↗

PubMed1992

Hormones increase oxygen uptake in periportal and pericentral regions of the liver lobule.

The effect of several hormones known to alter intracellular free Ca2+ on rates of O2 uptake in periportal and pericentral regions of the liver lobule was studied in the perfused liver. Regional O2 uptake was measured by stopping the flow and monitoring the decrease in O2 concentration. When perfusion was in the anterograde direction, basal rates of O2 uptake were two to three times higher in periportal than in pericentral regions, and phosphorylase alpha activity, which increases as a function of intracellular free Ca2+ levels, was higher in periportal regions. In contrast, when perfusion was in the retrograde direction, rates of O2 uptake were two to three times greater in pericentral regions. Infusion of epinephrine (0.1 microM) or angiotensin II (5 nM) increased the rate of O2 uptake nearly exclusively in downstream areas of the lobule where O2 tension was low. When perfusions were in the anterograde direction, epinephrine increased phosphorylase alpha activity significantly only in pericentral regions. Stimulation of O2 uptake by epinephrine was blocked by the alpha-adrenergic receptor blocker phentolamine (1 microM) but not by the beta-receptor blocker propranolol. Thus hormones that increase intracellular calcium stimulate O2 uptake predominantly in regions of the liver lobule where O2 tension is lowest, supporting the hypothesis that oxygen tension regulates O2 uptake in the liver via mechanisms involving intracellular free Ca2+.

Adrenergic alpha-Antagonists↗

PubMed1992

7-Ethoxycoumarin metabolism in hepatocytes from pre- and postpubescent male rats.

Marked changes in rates of drug metabolism occur during adolescence; however, biochemical events underlying alterations in drug metabolism in whole hepatocytes during this period of development are not well established. Accordingly, metabolism of 7-ethoxycoumarin, a model substrate for mixed-function oxidation, was studied in hepatocytes isolated from prepubescent and postpubescent male rats. Rates of 7-ethoxycoumarin O-deethylation increased 2.4-fold from 65 to 154 pmol/10(6) cells/min in intact hepatocytes during the narrow period of adolescence. In contrast, microsomal 7-ethoxycoumarin O-deethylase was the same in preparations from the two groups of animals. 7-Hydroxycoumarin glucuronide production in hepatocytes increased 2-fold and sulfate formation increased 16-fold across puberty. The results indicate that increases in drug metabolism, particularly sulfate conjugation, are mediated by biochemical events in addition to increases in total amounts and specific activities of hepatic drug-metabolizing enzymes.

Aging↗

PubMed1991

Inhibition of glucuronidation of benzo(a)pyrene phenols by long-chain fatty acids.

Long-chain fatty acids inhibit glucuronidation of benzo(a)pyrene phenols in perfused liver; therefore, this study was designed to investigate interactions of fatty acids with beta-glucuronidase, glucuronosyl transferase, and energy supply. In beta-glucuronidase-deficient C3H/He mice, infusion of oleate (250 microM) increased the release of free benzo(a)pyrene phenols from 14 to 33 nmol/g/h and decreased release of glucuronides into the perfusate from 25 to 17 nmol/g/h. Rates of accumulation of glucuronides in the liver were also diminished from 11 to 4 nmol/g/h after infusion of oleate (250 microM). Fatty acids did not affect the release of benzo(a)pyrene metabolites into bile, and the ratio of free phenol to glucuronide production was increased from 0.57 to 1.30. A similar trend was observed in livers from DBA/2 mice that have beta-glucuronidase. Rates of hydrolysis of benzo(a)pyrene-O-glucuronide were not altered in isolated microsomes by addition of oleoyl coenzyme A (CoA) or octanoyl CoA (10- approximately 100 microM). Thus, we conclude that fatty acids do not alter glucuronidation by acting on beta-glucuronidase. The concentration of cofactors (UDP-glucuronic acid, UDP-glucose, and adenine nucleotides) involved in hepatic conjugation was not altered by infusion of concentrations of oleate (300 microM) that inhibited glucuronidation in perfused livers. When oleate concentrations were increased to 600 microM, UDP-glucuronic acid and UDP-glucose decreased 44 and 49%, respectively, and the ATP:ADP ratio declined concomitantly. Oleoyl CoA inhibited UDP-glucuronosyl transferase noncompetitively (half-maximal inhibition, 10 microM) in microsomes with 3-hydroxy-benzo(a)pyrene or p-nitrophenol as substrate. In contrast, octanoyl CoA was a very poor inhibitor of transferase activity. Inhibition of the transferase by oleoyl CoA was increased markedly by treatment with detergents (Triton X-100), i.e., half-inhibition of glucuronosyl transferase was obtained with about 2 microM oleoyl CoA. Inhibition of UDP-glucuronosyl transferase by oleoyl CoA was also increased in a dose-dependent manner by albumin, possibly due to increasing access of the CoA derivative to the enzyme. Collectively, these data indicate that fatty acids diminish glucuronidation via the formation of acyl CoA compounds that inhibit UDP-glucuronosyl transferase noncompetitively.

Animals↗

PubMed1991

Effect of allyl alcohol on xanthine dehydrogenase activity in the perfused rat liver.

Xanthine oxidase has been implicated in the production of reactive oxygen species and cell injury produced by various toxic compounds. Since allyl alcohol injuries the liver by an oxygen-dependent mechanism, we examined the actions of this hepatotoxicant on the conversion of xanthine dehydrogenase into xanthine oxidase in perfused livers. A microassay for NAD(+)-dependent xanthine dehydrogenase, based on measuring the production of NADH fluorometrically under anaerobic conditions, was developed and used to examine the actions of allyl alcohol on this activity in periportal and pericentral regions of the liver lobule. The oxygen-dependent activity, xanthine oxidase, was monitored in whole liver homogenates by uric acid formation at 302 nm under aerobic conditions. Perfusion of the liver with allyl alcohol (350 microM) increased xanthine oxidase and decreased xanthine dehydrogenase in whole liver consistent with the hypothesis that allyl alcohol enhanced calcium-dependent proteolytic conversion of the NAD(+)-dependent to the O2-dependent form. Xanthine dehydrogenase was higher in pericentral than in periportal regions of the liver lobule and tended to decrease selectively in periportal zones of livers exposed to allyl alcohol. O2 uptake was stimulated transiently by allyl alcohol followed by subsequent inhibition of respiration. These results are consistent with the idea that conversion of NAD(+)-dependent xanthine dehydrogenase to xanthine oxidase is involved in the zone-specific hepatotoxicity of allyl alcohol.

1-Propanol↗

PubMed1991

Suppression of benzo[a]pyrene metabolism by accumulation of triacylglycerols in rat hepatocytes: effect of high-fat and food-restricted diets.

Diet has been implicated as a major determinant of chemical carcinogenesis. Accordingly, rates of benzo[a]pyrene (B[a]P) metabolism were compared in hepatocytes isolated from rats maintained on control, high-fat or food-restricted AIN-76A diets. Rats maintained on the food-restricted diet were given 65% of food consumed by the control group fed ad libitum. The high-fat diet group had free access to a modified AIN-76A diet in which the amount of corn oil was increased 4-fold at the expense of digestible carbohydrates. The triacylglycerol content in hepatocytes varied in direct proportion to dietary fat and calories and was 66 +/- 5, 105 +/- 7 and 192 +/- 16 nmol/mg dry wt in cells isolated from rats fed food-restricted, control and high-fat diets respectively. In contrast, the rate of B[a]P metabolism was highest in hepatocytes from rats maintained on the food-restricted diet and lowest in cells from animals given the high-fat diet (i.e. food-restricted greater than control greater than high-fat). Thus, an inverse correlation existed between the rate of B[a]P metabolism and the content of triacylglycerols in hepatocytes. At a cell density of approximately 2 mg dry wt/ml, rates of B[a]P (40 microM) metabolism were 1324 +/- 186, 1150 +/- 198 and 829 +/- 76 pmol/mg dry wt/h, respectively, in hepatocytes isolated from rats fed food-restricted, control and high-fat diets. When cells were incubated with a lower concentration of B[a]P (10 microM), the rate of B[a]P metabolism was greater than 2-fold higher in hepatocytes from rats fed the food-restricted diet compared to the rate measured in cells from the high-fat group. Glucuronidation of B[a]P metabolites in hepatocytes from rats fed high-fat diet was also approximately 30% lower than rates determined for control and food-restricted groups. These diet-induced alterations in rates of B[a]P metabolism occurred in the absence of changes in specific activity of arylhydrocarbon hydroxylase or UDP-glucuronosyltransferase in liver microsomes. Further, the rate of 7-ethoxycoumarin metabolism, a more hydrophilic substrate, was not affected by diet and B[a]P but not 7-ethoxycoumarin accumulated in hepatic lipid droplets. Thus, diet-induced changes in intracellular triacylglycerol, particularly in lipid droplets, may alter access of B[a]P to binding sites on arylhydrocarbon hydroxylase and thereby modulate B[a]P metabolism in intact hepatocytes.

Analysis of Variance↗

PubMed1991

The liver plays a central role in the mechanism of chemical carcinogenesis due to polycyclic aromatic hydrocarbons.

The important problem of whether metabolites and DNA adducts from benzo[a]pyrene (B[a]P) originate in the liver or target tissues was assessed using orthotopic liver transplantation. Following liver transplantation, the only source of metabolites for release into the blood and accumulation in target tissues is the liver. [3H]B[a]P (4 microM, 5 Ci/mmol) was infused into the portal vein of rats, and livers were perfused and either transplanted to a second rat or sham-operated and left in situ (non-transplant group). After 4 h, seven organs were collected and polar metabolites and DNA adducts were measured. In both groups, B[a]P in blood samples was below the limits of detection while levels of B[a]P in liver samples were approximately 5 pmol/g and polar metabolites were approximately 10 pmol/g. Concentrations of polar metabolites were also nearly identical in peripheral tissues from both groups. Phenols, glucuronides, sulfates and an unidentified metabolite of B[a]P were also similar, but GSH conjugate(s) had a tendency to be lower in blood of animals with transplanted livers. DNA adducts ranged from minimal values near levels of detection to approximately 0.2 pmol/mg DNA in lung, liver, and kidney. Importantly, there were no differences in DNA binding between the transplant and non-transplant groups. Taken together, these data provide compelling evidence that the liver is the predominant site of conversion of B[a]P into polar metabolites which are transported to target tissues and subsequently bind to DNA. Release of polar metabolites from the liver may represent a novel pathway for delivery of carcinogen conjugates to target tissues.

Animals↗

PubMed1991

Enzyme activities associated with carcinogen metabolism in liver and nonhepatic tissues of rats maintained on high fat and food-restricted diets.

The influence of high fat or food-restricted diets on key enzymes associated with polycyclic aromatic hydrocarbon metabolism was assessed in liver, lung, kidney and stomach of rats. Animals had access ad libitum to the AIN-76A purified diet (control) or were given 65% of the intake of controls for 3 wk. The high fat diet was isoenergetic to the control diet by substituting corn oil for equal energy from carbohydrate and addition of cellulose to obtain equal energy density. Activities of arylhydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase were significantly increased in lungs of food-restricted rats and decreased by the high fat diet but were not altered in liver. Both diets increased arylhydrocarbon hydroxylase approximately twofold in kidney. Glucose 6-phosphate and 6-phosphogluconate dehydrogenase were lowered in lung, kidney and liver by the high fat diet. Hepatic glutathione S-transferase was increased by high fat feeding. Food restriction decreased activities of arylsulfatase and beta-glucuronidase about 40% in lung. Hepatic arylsulfatase was also decreased about 40% by this treatment. Changes in hydrolase activities in livers and lungs of animals maintained on restricted diets raises in the interesting possibility that net production of glucuronide and sulfate conjugates of carcinogens by the liver and their hydrolysis in lung is altered by food restriction.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1991

Futile cycling of a sulfate conjugate by isolated hepatocytes.

The sulfate conjugate of the model compound 4-methylumbelliferone was taken up and hydrolyzed considerably more rapidly by isolated hepatocytes than was the glucuronide conjugate. Using intact hepatocytes or homogenates of hepatocytes, compounds were identified that either inhibited 4-methylumbelliferyl sulfate hydrolysis via arylsulfatase or impaired its uptake into cells. For example, sodium sulfate inhibited hydrolysis of 4-methylumbelliferyl sulfate by intact hepatocytes (half-maximal inhibition, 0.1 mM) but not by homogenates, suggesting a selective action on organic sulfate uptake at the plasma membrane. In contrast, cholesterol sulfate inhibited hydrolysis of 4-methylumbelliferyl sulfate by homogenates but not by hepatocytes, consistent with the hypothesis that cholesterol sulfate does not readily enter intact cells. Compounds that inhibited hydrolysis of 4-methylumbelliferyl sulfate by both isolated hepatocytes and microsomes include sodium sulfite (half-maximal inhibition, 0.1 mM), pregnenolone sulfate (half-maximal inhibition, 1 microM), and estrone sulfate (half-maximal inhibition, 10 microM). To test whether production of sulfate conjugates could be modified by agents affecting arylsulfatase in intact hepatocytes, we examined the effects of pregnenolone sulfate on the production of 4-methylumbelliferyl sulfate from 4-methylumbelliferone. Addition of pregnenolone sulfate (100 microM) to intact cells increased rates of 4-methylumbelliferone sulfate production and decreased the fraction of 4-methylumbelliferone converted into the glucuronide. Hydrolysis of 4-methylumbelliferyl sulfate by isolated microsomes was inhibited in a dose-dependent manner by adenosine 3'-phosphate 5'-phosphosulfate (PAPS) when cytosol, a source of sulfotransferase was present. Furthermore, addition of low concentrations of PAPS (0.5 microM) to a reconstituted system of microsomes and cytosol impaired the formation of fluorescent product from 4-methylumbelliferyl sulfate until PAPS was consumed, indicating that futile cycling via arylsulfatase and sulfotransferase occurred. Subsequent futile cycling of free 4-methylumbelliferone and 4-methylumbelliferyl sulfate occurred upon repeated additions of PAPS and was prevented by sodium sulfite, an inhibitor of arylsulfatase. These results argue strongly that sulfate conjugate production within hepatocytes is regulated by futile cycling via sulfotransferase and arylsulfatase. Thus, drugs and endogenous substances that affect arylsulfatase may have marked effects on sulfate conjugate production by the liver.

Animals↗

PubMed1990

Regulation of urea synthesis in sublobular regions of the liver lobule by oxygen.

Periportal and pericentral regions of the liver lobule were isolated from perfused rat liver using a micropunch and incubated in Krebs-Henseleit buffer (pH 7.6) containing 2% poly(ethylene glycol) in Eagle's basal medium, PMSF (50 micrograms/ml) and leupeptin (20 micrograms/ml) for 2 h at 25 degrees C under and O2/CO2 (95:5%) gas phase. Maximal rates of urea production from ammonium chloride were 96.4 +/- 8.7 and 32.8 +/- 5.4 mumol/g per h at 800 and 200 microM O2. Thus, urea synthesis was 2-3-times greater at high than low O2 tension in plugs from periportal and pericentral regions of the liver lobule.

Animals↗

PubMed1990

Interactions between plasticizers and fatty acid metabolism in the perfused rat liver and in vivo. Inhibition of ketogenesis by 2-ethylhexanol.

Rates of ketone body (beta-hydroxybutyrate plus acetoacetate) production by perfused livers from starved rats were decreased about 60% from 39 +/- 2 to 17 +/- 3 mumol/g/hr by 2-ethylhexanol (200 microM), a primary metabolite of the plasticizer diethylhexyl phthalate. Inhibition of ketogenesis by ethylhexanol was dose dependent (half-maximal inhibition occurred with 25 microM) in the presence or absence of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. Concentrations of beta-hydroxybutyrate relative to acetoacetate (B/A) increased in a step-wise manner from 0.32 to 0.75 in the effluent perfusate when ethylhexanol was infused. In contrast, the B/A ratio decreased in parallel with inhibition of ketone body production when alcohol dehydrogenase was inhibited. Pretreatment of rats with phenobarbital, an inducer of omega and omega-1 hydroxylases, diminished inhibition of ketone body production by low (less than 50 microM) of ethylhexanol. Thus, ethylhexanol is oxidized via phenobarbital-inducible pathways to metabolites which do not inhibit ketogenesis. Studies were conducted to determine the site of inhibition of fatty acid oxidation by ethylhexanol. Rates of ketone body production in the presence of oleate (250 microM), which requires transport of the corresponding CoA compound into mitochondria, were reduced from 80 +/- 6 to 58 +/- 8 mumol/g/hr by ethylhexanol. In contrast, ketone body production from hexanoate, which is activated in the mitochondria, was not affected by ethylhexanol. Basal and oleate-stimulated rates of H2O2 production were not affected by ethylhexanol, indicating that peroxisomal beta-oxidation was not altered by the compound. Based on these data it is concluded that 2-ethylhexanol inhibits beta-oxidation of fatty acids in mitochondria but not in peroxisomes. Treatment of rats with ethylhexanol (0.32 g/kg, i.p.) decreased plasma ketone bodies from 1.6 to 0.8 mM, increased hepatic triglycerides and increased lipid predominantly in periportal regions of the liver lobule. These data indicate that alterations in hepatic fatty acid metabolism in periportal regions of the liver lobule may be early events in peroxisome proliferation.

Animals↗

PubMed1990

Activation of glycogen phosphorylase in rat pheochromocytoma PC12 cells and isolated hepatocytes by organophosphates.

Several organophosphates including diisopropylfluorophosphonate (DPF) and a variety of compounds used as chemical warfare agents produced dose- and time-dependent increases in phosphorylase-a, the phosphorylated form of glycogen phosphorylase in rat pheochromocytoma cells, PC12, and isolated hepatocytes. Increases in phosphorylase-a did not occur in cells exposed to the carbamates, physostigmine or pyridostigmine, or to O-ethyl S-2-diisopropylaminoethylmethyl-phosphonathiolate (VX), an organophosphate which is protonated at physiological pH. When extracellular pH was increased to pH 8, VX acted like the other organophosphates and increased phosphorylase-a activity. The possibility that organophosphates increase phosphorylase-a in intact cells by releasing Ca2+ from intracellular binding sites is supported by the following findings: organophosphate-induced increases in phosphorylase-a did not correlate with changes in cyclic AMP in the two cell types studied; in PC12 cells, increases in this activity occurred in the absence of extracellular calcium and were not inhibited by the calcium channel blocker, verapamil; fluorescence of the calcium sensitive dye, Quin-2, in PC12 cells preloaded with the acetoxymethyl ester of the dye was increased by soman; finally, addition of the calcium ionophore, A23187, to PC12 cells maintained in calcium-free medium caused sarin-stimulated phosphorylase-a activity to return rapidly to basal levels. Collectively, these data argue strongly that organophosphates increase phosphorylase-a activity in intact cells via a novel mechanism involving release of calcium from intracellular binding sites.

Adenine Nucleotides↗

PubMed1990

Effect of epinephrine on glycogen phosphorylase-alpha in various preparations of rat liver.

1. Glycogen phosphorylase-alpha, a commonly used index of cytosolic free calcium, was compared in various preparations of rat liver in the absence and presence of 0.1 microM epinephrine. 2. Total phosphorylase in isolated perfused livers and freshly-isolated hepatocytes were the same as that observed in liver in situ; however, phosphorylase-alpha was 50% higher in perfused liver and 80% higher in hepatocytes than activities measured in situ. Total phosphorylase was reduced approximately 50% in hepatocytes maintained in primary culture for 24 hr. 3. Epinephrine increased phosphorylase-alpha approximately 2-fold in livers perfused for 30 min but only about 20% in hepatocytes incubated for 30 min. After 90 min of perfusion or incubation, epinephrine increased phosphorylase-alpha nearly 4-fold in perfused livers and only 30% in isolated hepatocytes. The results suggest that amounts of free calcium and calcium-dependent coupling of adrenergic receptors to phosphorylase-alpha differ markedly between the intact liver and isolated hepatocytes.

Animals↗