Brian G. Lake

Scaling Factors for the Extrapolation of In Vivo Metabolic Drug Clearance From In Vitro Data: Reaching a Consensus on Values of Human Micro-somal Protein and Hepatocellularity Per Gram of Liver

Reported predictions of human in vivo hepatic clearance from in vitro data have used a variety of values for the scaling factors human microsomal protein (MPPGL) and hepatocellularity (HPGL) per gram of liver, generally with no consideration of the extent of their inter-individual variability. We have collated and analysed data from a number of sources, to provide weighted meangeo values of human MPPGL and HPGL of 32 mg g-1 (95% Confidence Interval (CI); 29-34 mg.g-1) and 99x10(6) cells.g-1 (95% CI; 74-131 mg.g-1), respectively. Although inter-individual variability in values of MPPGL and HPGL was statistically significant, gender, smoking or alcohol consumption could not be detected as significant covariates by multiple linear regression. However, there was a weak but statistically significant inverse relationship between age and both MPPGL and HPGL. These findings indicate the importance of considering differences between study populations when forecasting in vivo pharmacokinetic behaviour. Typical clinical pharmacology studies, particularly in early drug development, use young, fit, healthy male subjects of around 30 years of age. In contrast, the average age of patients for many diseases is about 60 years of age. The relationship between age and MPPGL observed in this study estimates values of 40 mg.g-1 for a 30 year old individual and 31 mg.g-1 for a 60 year old individual. Investigators may wish to consider the reported covariates in the selection of scaling factors appropriate for the population in which estimates of clearance are being predicted. Further studies are required to clarify the influence of age (especially in paediatric subjects), donor source and ethnicity on values of MPPGL and HPGL. In the meantime, we recommend that the estimates (and their variances) from the current meta-analysis be used when predicting in vivo kinetic parameters from in vitro data.

Studies on the hepatic effects of orally administered di-(2-ethylhexyl) phthalate in the rat

A sequential study of hepatic effects was conducted in young male Wistar albino rats following daily oral intubation of di-(2-ethylhexyl) phthalate (DEHP) at a dose of 2000 mg/kg for 21 days. The relative liver weights of the animals increased progressively with the duration of the treatment. Alcohol dehydrogenase activity and microsomal protein and cytochrome P-450 contents showed a marked initial increase followed by a reversal as the treatment progressed. In contrast to this biphasic response, the activities of microsomal glucose-6-phosphatase, aniline 4-hydroxylase, and mitochondrial succinate dehydrogenase activity were significantly depressed, as observed both biochemically and histochemically, throughout the period of exposure. Parallel electronmicroscopic studies revealed a progressive dilatation of the smooth and rough endoplasmic reticulum, mitochondrial swelling and an increase in microbodies. Investigations carried out on the metabolic fate of [14C]DEHP in animals prior to and during the course of DEHP treatment showed no significant differences in the excretion pattern of radioactivity. Furthermore, there was no evidence indicating the storage of phthalate residues in the liver. Studies on the comparative effects of phthalic acid, 2-ethylhexanol, and mono-(2-ethylhexyl)phthalate (MEHP) orally administered at dose levels equimolar to DEHP for 7 days showed that the biochemical and ultrastructural changes in the hepatic endoplasmic reticulum and mitochondria in DEHP pretreatment were substantially reproducible by the administration of MEHP. Additionally, it was found that 2-ethylhexanol treatment led to an increase in the number of microbodies. The results indicate that the partial hydrolysis of DEHP to the monoester (MEHP) is the degradative step which determines the hepatic changes produced by DEHP.

Peroxisome proliferation in primary cultures of rat hepatocytes

Primary cultures of rat hepatocytes were exposed to a range of chemicals known to cause peroxisome proliferation in vivo. Peroxisomal palmitoyl-CoA oxidation and carnitine acetyltransferase (CAT) activity were increased within 12 to 24 hr of adding 0.5 mM clofibrate to the culture medium, reaching about 20 times control levels after 72 hr. Stimulation of CAT activity was dose related over a concentration range of 0.05 to 2 mM clofibrate and 0.02 to 0.2 mM mono-2-ethylhexylphthalate (MEHP). Higher concentrations of MEHP were cytotoxic. The stimulation of CAT activity and palmitoyl-CoA oxidation produced by clofibrate and MEHP was inhibited by cycloheximide. In further studies with clofibrate and a range of other known peroxisome proliferators (nafenopin, tiadenol, BR-931, Wy-14,643, and acetylsalicylic acid), induction of CAT and palmitoyl-CoA oxidation was observed with no increase in activity of another peroxisomal enzyme, D-amino acid oxidase. This differential effect on peroxisomal enzyme activity is typical of that seen in vivo. Furthermore, the relative potencies of the different peroxisome proliferators in vitro agreed well with what is known from studies in vivo. Mitochondrial and microsomal marker enzymes showed little change in activity. Electron microscopy of treated cultures revealed increased numbers of peroxisomes, some of which lacked the characteristic nucleoid. The results indicate that primary cultures of rat hepatocytes provide a rapid, sensitive means of identifying chemicals that cause peroxisome proliferation and a potentially valuable system for studies aimed at clarifying the toxicological significance of this phenomenon.

Microsomal cytochrome P-452 induction and peroxisome proliferation by hypolipidaemic agents in rat liver: A mechanistic inter-relationship

Eight structurally diverse hypolipidaemic agents have been examined for their ability to induce the microsomal cytochrome P-452-dependent fatty acid hydroxylase system and the enzymes of peroxisomal beta-oxidation in rat liver. Using a specific ELISA method, we have shown that the cytochrome P-452 isoenzyme is induced up to ten fold by hypolipidaemic challenge, concomitant with a pronounced elevation of the peroxisomal beta-oxidation enzymes, mirrored by an increase in peroxisomal volume as determined morphometrically. In addition, the induction of cytochrome P-452 is accompanied by a decrease in the activities of cytochromes P-450b and P-450c as measured by benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities respectively, the latter being more extensively reduced by hypolipidaemic treatment. A hypothesis is presented whereby an early biological response is the hypolipidaemic induction of microsomal cytochrome P-452 resulting in omega-hydroxy fatty acids and their subsequent further oxidation to dicarboxylic acids, the latter providing the proximal stimulus for peroxisomal proliferation.

The in vitro hydrolysis of some phthalate diesters by hepatic and intestinal preparations from various species

A study was made of the hydrolysis of dimethyl, diethyl, di-n-butyl, di-n-octyl, di-(2-ethylhexyl), and dicyclohexyl phthalates by both hepatic and intestinal preparations from various species. Hepatic preparations from the rat, baboon, and ferret hydrolyzed each of the phthalate diesters to their corresponding monoester derivatives. Additionally, intestinal preparations from the three animal species and from man also catalyzed the monohydrolysis of phthalate diesters. These results thus show a species similarity in the metabolism of phthalate diesters between man, a rodent, a nonrodent, and a nonhuman primate species. Furthermore, the results suggest that orally ingested phthalate diesters would most probably be absorbed from the gut of the rat, baboon, ferret, and man primarily as the corresponding monoester derivatives. Hence, any toxic effects of orally administered phthalate diesters would be governed by the properties of the constituent phthalate monoesters and/or alcohols.

Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator

Abstract The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

Comparative studies on nafenopin-induced hepatic peroxisome proliferation in the rat, Syrian hamster, guinea pig, and marmoset☆

Nafenopin was administered orally for 21 days to male Sprague-Dawley rats (0.5-50 mg/kg/day), Syrian hamsters (5-250 mg/kg/day), Dunkin-Hartley guinea pigs (50 and 250 mg/kg/day), and marmosets (Callithrix jacchus, 50 and 250 mg/kg/day). With the rat, and to a lesser extent in the hamster, nafenopin treatment produced dose-related increases in liver size and induction of peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidizing enzyme activities. In contrast, in the guinea pig and marmoset, there was no effect on liver size and only comparatively small changes were observed in these enzyme activities. Ultrastructural examination of liver sections from nafenopin-treated rats and hamsters revealed increased numbers of peroxisomes many of which lacked the characteristic crystalline nucleoid. While nafenopin had little effect on peroxisome numbers in either the guinea pig or marmoset, increases in microsomal cytochrome P450 content and mixed function oxidase activities were observed in these species. These results demonstrate marked species differences in nafenopin-induced hepatic peroxisome proliferation with the Syrian hamster being less responsive than the rat and the guinea pig and marmoset being only weakly responsive. As nafenopin is a known hepatocarcinogen in the rat, comparative long-term studies in poorly responsive species, such as the guinea pig and marmoset, may help clarify the role of organelle proliferation in the hepatocarcinogenicity of certain peroxisome proliferators.

Peroxisome proliferation in cultured rat hepatocytes produced by clofibrate and phthalate ester metabolites

Adult rat hepatocytes cultured for 48 h in the presence of 0.2 mM clofibrate, mono-(2-ethylhexyl)-phthalate (MEHP) or 2-ethylhexanol (2-EHA) contained increased numbers of peroxisomes. In keeping with the effects of these compounds in vivo, the peroxisome proliferation was associated with marked increases (up to 15-fold) in the activity of carnitine acetyltransferase. No such effects were produced by n-hexanol or two microsomal enzyme inducers, phenobarbital and 1,2-benzanthracene. These results suggest that cultured hepatocytes may provide a useful model system for studying chemically induced peroxisome proliferation.

Induction of drug metabolism: Species differences and toxicological relevance

A large number of drugs and other chemicals have been shown to induce hepatic microsomal cytochrome P450 (CYP) forms in experimental animals and humans. Most CYP forms are induced by receptor-mediated mechanisms leading to an increase in gene transcription. Important nuclear receptors involved in the induction of CYP1A, CYP2B, CYP3A and CYP4A subfamily forms comprise, respectively, the aryl hydrocarbon receptor, the constitutive androstane receptor, the pregnane X receptor and the peroxisome proliferator-activated receptor alpha. Hepatic CYP form induction can be assessed by in vivo, ex vivo and in vitro methods. Significant species differences can exist in the enzyme induction response to a given chemical and also in the toxicological consequences of induction. Hepatic CYP form induction in humans may lead to clinically important drug-drug interactions. In rodents hepatic CYP form induction can be associated with the formation of tumours by non-genotoxic modes of action in the liver, thyroid and other tissues.

Induction of cytochrome P-450-dependent enzyme activities in cultured rat liver slices

Precision-cut liver slices were prepared from male Sprague-Dawley rats with a Krumdieck tissue slicer and cultured in RPMI 1640 medium for up to 72 hr. After 48 hr, cytochrome P-450 content in the slices declined to 36% of levels present in freshly cut rat liver slices. The addition of either beta-naphthoflavone (BNF) or Aroclor 1254 (ARO) partially prevented the loss of cytochrome P-450. Culture of liver slices with phenobarbitone (PB), BNF and ARO resulted in the induction of 7-ethoxycoumarin O-deethylase, 7-benzoxyresorufin O-debenzylase and 7-ethoxyresorufin O-deethylase activities. Generally, the induction of mixed-function oxidase enzymes was greater in 72- than in 48-hr cultured slices, and at the concentrations examined ARO produced a greater stimulation of enzyme activities than did either PB or BNF. These results demonstrate that rat liver slices may be maintained in culture for up to 72 hr, and that they respond in a similar manner to rat primary hepatocyte cultures to some inducers of xenobiotic metabolism. Precision-cut liver slices may therefore be a useful alternative in vitro system to hepatocyte cultures for screening compounds for effects on mixed-function oxidases and for assessing species differences in response.