Loss Of Cytochrome P-450 Content Research Articles

Inhibition of cytochrome P450 2E1 and activation of transcription factor Nrf2 are renoprotective in myoglobinuric acute kidney injury

Rhabdomyolysis accounts for ∼10% of acute kidney injuries. In glycerol-induced myoglobinuric acute kidney injury, we found an increase in the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear protein, a key redox-sensitive transcription factor, and Nrf2-regulated genes and proteins including upregulation of heme oxygenase-1. In in vitro studies, pretreatment of LLC-PK1 cells with an activator of Nrf2 before myoglobin exposure significantly decreased oxidant generation and cytotoxicity, whereas Nrf2 inhibition and gene silencing exacerbated the injury. Chlormethiazole, a specific CYP2E1 transcription inhibitor, prevented an increase in catalytic iron in the kidneys, decreased oxidative stress, blocked nuclear translocation of the Nrf2 protein, decreased heme oxygenase-1 upregulation, and provided functional and histological protection against acute kidney injury. CYP2E1 inhibitors and gene silencing in renal tubular epithelial cells significantly decreased reactive oxygen species generation and provided marked protection against myoglobin-induced cytotoxicity. Thus, during CYP2E1-induced oxidative stress, the transcription factor Nrf2 has a pivotal role in the early adaptive response. Inhibition of CYP2E1 coupled with the prior induction of Nrf2 may be a valuable tool to reduce CYP2E1-mediated rhabdomyolysis-induced acute kidney injury.

Activation of signalling pathways during hepatocyte isolation: relevance to toxicology in vitro

The “Holy Grail” of in vitro toxicology is to develop assay systems that mimic the in vivo situation and hence reduce the need for toxicity tests employing experimental animals. However a major problem to be overcome with cell culture models is the rapid loss of differentiated phenotype that markedly limits extrapolation of results to the whole animal (i.e. human) situation. This limitation is most obvious in the application of hepatocyte cultures to predict pathways of metabolism mediated toxicity and results from the rapid loss of cytochrome P450 content. Here we demonstrate that changes in hepatocyte gene expression (e.g. MAP kinase and NF-κB activation) occur very early into the well established hepatocyte isolation procedure employing collagenase suggesting that hepatocytes are undergoing a pro-inflammatory (‘acute phase’) response before they are cultured. Data is presented indicating that the stimulus is, in part, due to oxidative stress but the demonstration of endotoxins in collagenase preparations is likely to exacerbate the situation. Thus appreciation of these early events during hepatocyte isolation represents the surest foundation for the successful application of cultured hepatocytes to toxicology rather than relying on traditional manipulations of hepatocyte culture medium/substratum once differentiated phenotype has already been lost.

Use of Primary Rat and Human Hepatocyte Sandwich Cultures for Activation of Indirect Carcinogens: Monitoring of DNA Strand Breaks and Gene Mutations in Co-cultured Cells

Loss of cytochrome P-450 content is a common feature in conventional culture systems of primary hepatocytes. In contrast to the standard in vitro situation, in vivo each hepatocyte is exposed to an extracellular matrix (space of Disse) at two opposing basolateral surfaces. This in vivo symmetry has been reconstructed in vitro by culturing rat or human hepatocytes within two layers of collagen, thus forming a sandwich configuration. Activation of dimethylbenzanthracene (DMBA) or benzo[ a]pyrene (BaP) was studied in rat and human hepatocytes. Genotoxic effects were studied in a three-dimensional co-culture model between sandwich hepatocytes and mammalian cells using the comet assay for detection of DNA strand breaks, and the HPRT test for detection of gene mutations. Sandwich hepatocytes generated active metabolites. The maintenance of metabolic properties in hepatocytes was dependent on extracellular matrix geometry. The number of DMBA- or BaP-induced genotoxic effects tended to be higher than in standard S-9 mix assays. While the ability to activate indirect carcinogens disappears within hours in primary hepatocytes, hepatocyte sandwich cultures enhance their ability to activate indirect carcinogens within 1 wk and retain this activity for up to 2 wk. This is the main advantage of the sandwich method over the more simple and conventional assays. While freshly isolated hepatocytes, regardless of whether in sandwich culture or in conventional assays, are injured by the isolation procedure and possess a corresponding reduced activation ability, hepatocytes in sandwich cultures recover over the course of a few days, and acquire a much higher ability to activate indirect carcinogens. Consequently, the indirect carcinogens BaP and DMBA, which were ineffective (BaP) or exhibited only weak effects (DMBA) at a concentration of 160 nmol/ml in 1–2-day-old hepatocytes, were clearly effective (BaP) or showed about a threefold increase in genotoxicity (DMBA) in 8-day-old hepatocytes in sandwich co-culture. In contrast to the experiments with S-9 mix, which is toxic to mammalian cells and does not allow treatment times of more than 2–3 hr, cells in co-culture with human or rat hepatocytes can be treated for at least 24 hr. The use of sandwich cultures has not yet been described for genotoxicity studies. The results of the present study may perhaps facilitate the acceptance of this method as a co-culture model for the field of genetic toxicology. Use of hepatocytes alone for genotoxicity studies cannot be recommended for difficulties in isolating intact cells from the sandwich cultures. The use of human hepatocytes in sandwich co-culture should enable a more relevant evaluation of potential human genotoxicity with specific chemicals and should put the extrapolation of genetic toxicology data from animal species to humans on a more scientific basis. Beyond that, experiments with animals in vivo could be avoided.

Evidence for cytochrome P-450 as a source of catalytic iron in myoglobinuric acute renal failure

Iron has been implicated to play an important role in several models of tissue injury, including myoglobinuric acute renal failure. In this model, myoglobin released from the injured muscle is generally accepted as a source of iron. In the present study we measured the bleomycin-detectable iron (iron capable of catalyzing free radical reactions) in the kidneys and examined the role of cytochrome P-450 as a source of catalytic iron in glycerol-induced model of myoglobinuric acute renal failure. Rats were injected with 50% glycerol (8 ml/kg) i.m. after overnight water deprivation and sacrificed 24 hours later. There was a marked and a specific increase in the bleomycin-detectable iron content accompanied by a marked decrease in the cytochrome P-450 content in the kidneys of glycerol treated rats. We then examined the effects of two different cytochrome P-450 inhibitors, cimetidine (with ranitidine as a control) and piperonyl butoxide. Cimetidine, but not ranitidine, significantly prevented the increase of bleomycin-detectable iron in the kidneys of glycerol-treated rats. The loss of cytochrome P-450 content was substantially blocked by both inhibitors, cimetidine and piperonyl butoxide, but not by ranitidine. Both the inhibitors of cytochrome P-450 provided functional (as measured by BUN and creatinine) and histological protection against glycerol-induced acute renal failure. Our data thus demonstrate a marked increase in bleomycin-detectable iron in the kidneys of glycerol-treated rats. Our data also indicate that inhibitors of cytochrome P-450 provide protection against glycerol-induced acute renal failure and that cytochrome P-450 may be a significant source of this iron in this model of acute renal failure.

Inhalation pharmacokinetics of carbon tetrachloride in rats based on arterial blood:Inhaled air concentration ratios

To estimate the rate of CCl 4 metabolism in vivo by using an inhalation pharmacokinetic approach based on arterial blood:air concentration ratios, the blood CCl 4 concentrations ( C art) at the end of 5-hr exposure to varying concentrations of CCl 4 in inhaled air ( C inh) were determined in male, naive rats and in rats pretreated with po administration of 100 or 200 μl CCl 4/100 g body weight 24 hr before exposure. Hepatic cytochrome P-450 content during and at the end of exposure was also determined. The biphasic nature of the C art- C inh curve for naive rats, with a transition at C inh of about 100 ppm, indicated that CCl 4 metabolism is perfusion-limited below 100 ppm and is saturated above 100 ppm. In 100 μl CCl 4-pretreated rats, C inh at the transition point decreased from 100 to 50 ppm; this percentage decrease was consistent with the decreased cytochrome P-450 content induced by administration of 100 μl CCl 4. In 200 μl CCl 4-pretreated rats, where CCl 4 metabolizing enzyme activity was completely inhibited, the C art- C inh curve gave a single line with a shallower slope than that of the steeper part of the curve for naive rats, reflecting a loss of cytochrome P-450 content during exposure. The curves of calculated uptake rate showed continued uptake in completely inhibited rats, representing the contribution of fat loading only. The rate of metabolism was approximated by the uptake rate for naive rats minus that for 200 μl CCl 4-pretreated rats, and decreased gradually with increasing C inh over the range of saturable metabolism. From this rate curve, V max and K m for naive rats were 2.7 mg/kg/hr and of the order of 0.3 mg/liter, respectively. The gradual decrease in the rate of metabolism could be interpreted in terms of the rapid loss of cytochrome P-450 content. The V max for 100 μl CCl 4-pretreated rats decreased by about 57%, which was in good agreement with the decrease of cytochrome P-450 content. These experiments suggest the usefulness and validity of this approach for studying metabolism of a volatile compound.

Depression of Hepatic Microsomal Enzyme Systems by Lentinan in Mice

Studies were performed to determine the effects of an immunopotentiating agent, lentinan, on the hepatic drug-metabolizing enzymes in mice. Lentinan was injected twice a day for two days, and the enzyme activities were determined 12 hr after the last injection of lentinan. A lentinan dose of over 0.25 mg/kg was required to cause a significant decrease (20–40%) in the hepatic microsomal aminopyrine N-demethylase and aniline hydroxylase activities. The loss of drug-metabolizing activity by the treatment with lentinan agreed with the loss of cytochrome P-450 content in many cases. Strain and substrate differences concerning the effect of lentinan on the metabolism of drug were also observed. That is to say, the loss of cytochrome P-450 content by the treatment with lentinan was observed in the ddY, C57BL/6 and BDF1 strain mice, but was not observed in the DBA/2, C3H/He and C57BL/10 strain mice. The decrease in the activities of 7-ethoxycoumarin O-deethylase and biphenyl 2-hydroxylase by the treatment with lentinan was considerably less than that of aminopyrine N-demethylase and aniline hydroxylase in ddY mice.

Protective action of diethyldithiocarbamate and carbon disulfide against renal injury induced by chloroform in mice

Oral administration of diethyldithiocarbamate (DTC) and carbon disulfide (CS 2) protected mice against CHCl 3-induced kidney injury, as evidenced by normalization of delayed plasma phenolsulfonphthalein clearance, suppression of increased kidney calcium content and prevention of renal tubular necrosis. In CCl 4-treated mice, in which liver microsomal monooxygenase activities were decreased markedly, and kidney microsomal aniline hydroxylase and p-nitroanisole demethylase activities were increased to about twice those of the untreated mice, renal toxicity of CHCl 3 was greatly potentiated, and the latter effect was also blocked by both agents. DTC and CS 2 per se markedly decreased kidney microsomal aniline hydroxylase and p-nitroanisole demethylase activities at 1 hr after oral administration, accompanying a moderate loss of cytochrome P-450 content, in both normal and CCl 4-treated mice. The protection was not due to hypothermia, because pretreatment with DTC or CS 2 (p.o.) also prevented the hypothermia induced by CHCl 3. The mechanism of the protection may have involved inhibition of metabolic activation of CHCl 3 in the kidney rather than in the liver.

EFFECT OF LINOLEIC ACID HYDROPEROXIDEON LIVER MICROSOMAL ENZYMES IN VITRO

Rat liver microsomes incubated with linoleic acid hydroperoxide (LAHPO) lost cytochrome P-450 specifically among the enzymes of microsomal electron transport systems. The loss of cytochrome P-450 content and glucose-6-phosphatase activity by LAHPO was accompanied by an increase in malondialdehyde (MDA) production. Turbidity of microsomal suspensions was decreased with increasing MDA production, but not proportionately. Diethyldithiocarbamate (DTC), N,N'-diphenyl-p-phenyl- enediamine and α-tocopherol inhibited almost completely the LAHPO-induced MDA production of microsomes, however no perfect protection against the loss of cytochrome P-450 content and glucose-6-phosphatase activity was observed. The decrease of microsomal turbidity by LAHPO was little affected in the presence of DTC. Purified cytochrome P-450 was destroyed by LAHPO, with minimal protection by the compounds described above. These results suggest the possibility that the loss of microsomal enzyme activities during lipid peroxidation may be attributed largely to a direct attack on enzyme proteins by lipid peroxides rather than indirectly to a structural damage of microsomal membranes resulting from peroxidative breakdown of membrane lipids.