M6P/IGF2R gene is mutated in human hepatocellular carcinomas with loss of heterozygosity.

The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) functions in the intracellular trafficking of lysosomal enzymes, the activation of the potent growth inhibitor, transforming growth factor beta 2, and the degradation of IGF2 (ref. 1), a mitogen often overproduced in tumours. We have recently shown that 70% of human hepatocellular tumours have loss of heterozygosity (LOH) at the M6P/IGF2R locus which maps to chromosome 6q26-q27 (ref. 8). Using a coarse screen, we have now identified point mutations in the remaining allele of 25% of human hepatocellular carcinomas (HCCs) with LOH. These mutations give rise to truncated receptor protein and significant amino acid substitutions, and provide evidence that the M6P/IGF2R gene functions as a tumour suppressor in human liver carcinogenesis.

Statin-induced myopathy in the rat: relationship between systemic exposure, muscle exposure and myopathy.

Rare instances of myopathy are associated with all statins, but cerivastatin was withdrawn from clinical use due to a greater incidence of myopathy. The mechanism of statin-induced myopathy with respect to tissue disposition was investigated by measuring the systemic, hepatic, and skeletal muscle exposure of cerivastatin, rosuvastatin, and simvastatin in rats before and after muscle damage. The development of myopathy was not associated with the accumulation of statins in skeletal muscle. For each statin exposure was equivalent in muscles irrespective of their fibre-type sensitivity to myopathy. The low amount of each statin in skeletal muscle relative to the liver does not support a significant role for transporters in the disposition of statins in skeletal muscle. Finally, the concentration of cerivastatin necessary to cause necrosis in skeletal muscle was considerably lower than rosuvastatin or simvastatin, supporting the concept cerivastatin is intrinsically more myotoxic than other statins.

Frequent loss of heterozygosity on 6q at the mannose 6-phosphate/insulin-like growth factor II receptor locus in human hepatocellular tumors.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIr) is required for the activation of transforming growth factor beta, and previously we have found its expression to be significantly reduced in both rat and human hepatocellular carcinomas (HCCs). Therefore, we have postulated that loss of the M6P/IGFIIr gene may be mechanistically involved in liver carcinogenesis. Using the polymerase chain reaction, we utilized two polymorphisms in the 3' untranslated region of the M6P/IGFIIr gene to screen non-cirrhotic, hepatitis virus negative patients with hepatocellular tumors for LOH. Twenty-two of 36 (61%) patients were informative (heterozygous), and 14/22 (64%) liver tumors had LOH; 11/16 (69%) carcinomas, 1/3 (33%) fibrolamellar tumors and 2/3 (67%) adenomas. This is the first report of LOH at the M6P/IGFIIr locus in human hepatocellular tumors, and the presence of LOH in adenomas suggests that allelic loss may be an early event in the etiology of HCCs. These results support the hypothesis that the M6P/IGFIIr gene may function as a tumor suppressor gene in the liver.

The physiological disposition of 14 C-methsuximide in the rat.

1. (14)C-Methsuximide (N-methyl-(14)C-2-methyl-2-phenylsuccinimide) was rapidly absorbed from the small intestine of the rat (t(1/2) 17.4 min).2. The drug was rapidly and fairly evenly distributed throughout the body with peak blood and tissue levels occurring 1 h after oral administration. At all times, adrenals, body fat, kidneys and liver had higher levels of (14)C-methsuximide than other tissues and the drug freely traversed the blood-brain barrier. However, radioactivity disappeared rapidly from most tissues after the initial phase of the distribution.3. During 24 h, 26% of the orally administered radioactivity was recovered in urine and 29% appeared in expired air as (14)CO(2). The excretion of (14)CO(2) indicated N-demethylation of (14)C-methsuximide to 2-methyl-2-phenylsuccinimide. 2.7% of an administered dose of methsuximide was excreted unchanged in 24 h urine and 2.7% appeared as 2-methyl-2-phenylsuccinimide. 2-Methyl-2-phenylsuccinimide was also detected as a urinary metabolite of methsuximide in man.4. 2-Methyl-2-phenylsuccinimide possesses anticonvulsant activity and it is suggested that this metabolite contributes to the overall anticonvulsant activity and toxicity of methsuximide.5. Rat urine also contained a radioactive substance with similar chromatographic properties to one of the products of alkaline hydrolysis of methsuximide. This compound may arise from the spontaneous decomposition of the parent drug in vivo.

Hepatic microsomal glucuronidation of clofibric acid in the adult and neonate albino rat.

Hepatic microsomal UDP glucuronyltransferase activity towards the acid substrate clofibric acid has been described in the adult and neonate albino rat. The enzyme was maximally activated, approximately 2-fold, in the presence of 0.1-0.4% (w/v) digitonin. Induction of the digitonin activated clofibric acid glucuronyltransferase was observed following phenobarbitone treatment in vivo (2.2-fold), and to a lesser extent, following beta-naphthoflavone treatment (1.3-fold). Clofibrate treatment in vivo (of which clofibric acid is the ester hydrolysis product) had no effect on clofibric acid glucuronidation in vitro. The activity of clofibric acid glucuronyltransferase in the liver of rat before and at birth was low (approx. 0.08 nmoles glucuronide formed/min/mg microsomal protein). The activity increased 5-fold during the first three post-natal days. After this time, the activity increased linearly reaching adult levels by four weeks after birth. The data indicated that clofibric acid glucuronyltransferase belongs to the neonatal cluster of enzymes and clofibric acid is a group 2 substrate. Clofibric acid, a common therapeutic agent, is a useful, acid substrate for the estimation of mammalian hepatic microsomal glucuronyltransferase activity.

Comparative pharmacokinetics and subacute toxicity of di(2-ethylhexyl) phthalate (DEHP) in rats and marmosets: extrapolation of effects in rodents to man.

Certain phthalate esters and hypolipidemic agents are known to induce morphological and biochemical changes in the liver of rodents, which have been associated with an increased incidence of hepatocellular tumors in these species. There is evidence that hypolipidemic agents do not induce these effects in either subhuman primates or man. The oral and intraperitoneal administration of di(2-ethylhexyl) phthalate (DEHP) to the marmoset monkey at doses up to 5 mmole DEHP/kg body weight/day for 14 days did not induce morphological or biochemical changes in the liver or testis comparable with those obtained in rats given the same amount of DEHP. In the marmoset, the excretion profile of [14C]-DEHP following oral, IP, and IV administration and the lower tissue levels of radioactivity demonstrated a considerably reduced absorption in this species compared to the rat. The urinary metabolite pattern in the marmoset was in many respects qualitatively similar to but quantitatively different from that in the rat; the marmoset excreted principally conjugated metabolites derived from omega- 1 oxidation. The pharmacokinetic differences between these two species indicate that the tissues of the marmoset are exposed to a level of DEHP metabolites equivalent to the complete absorption of a dose of Ca. 0.1 to 0.25 mmole DEHP/kg body weight/day without significant toxicological effects. These exposure levels are at least 100-fold greater than the worst estimates of incidental human exposure (ca. 0.0015 mmole/kg/day). They are comparable with the human therapeutic dose of many hypolipidemic drugs (ca. 0.15 mmole/kg/day), a dose at which it is claimed that there is an absence of morphological or biochemical changes to human or subhuman primate liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic activation and bacterial mutagenicity of aflatoxin B1 in two different test systems.

The metabolism of aflatoxin B1 (AFB1) by liver post-mitochondrial supernatant fraction from phenobarbitone treated male albino rats in liquid suspension and soft-agar plate incorporation bacterial mutation assay systems has been studied. The AFB1 concentration used was in the range of those added to bacterial mutagenicity tests with this mycotoxin. Three oxidative metabolites of AFB1 viz the Tris derivative of AFB1 8,9-diol (derived from the 8,9-epoxide), aflatoxin Q1 (AFQ1) and M1 (AFM1) were observed. The metabolite profile and time course of formation were qualitatively similar in both assay systems. The rate and overall formation of metabolites in the soft-agar system was approximately one half that in the liquid suspension system which was reflected in decreased AFB1 induced bacterial mutation in the former system. The AFB1 metabolite profile in these in vitro systems did not mirror completely the reported in vivo profile seen in the rat, as aflatoxin P1 (AFP1) and a glutathione conjugate were not detected. No evidence was found for a prolongation of metabolism in the soft-agar system.

Metabolic capability of rat hepatocytes isolated by perfusion and tissue slice techniques.

The metabolic capability of hepatocytes prepared by the perfusion method (P cells) and the tissue slice method (S cells) has been compared using standardised procedures. Yields of P cells were four times greater than for S cells. Trypan blue exclusion viability and oxygen utilization were similar although the viability of S cells deteriorated faster with time. P cells had a lower endogenous rate of glycogenolysis and showed better glucagon stimulation than S cells. Similarly, P cells performed gluconeogenesis at a higher rate. However, there was no significant differences in the metabolism of the xenobiotic ethoxycoumarin. It is concluded that while S cells are probably satisfactory for studies of drug metabolism their use for work involving surface receptor binding and energy demanding processes should be questioned.

Localization and identification of cytochrome P-450 in differentiating rat embryo cells in vitro and in foetal tissue in vivo by immunocytochemistry.

The presence of the isoenzymes b, e and c of cytochrome P-450 in foetal rat limb-bud and mid-brain tissue has been investigated in vivo and in micromass cell cultures of limb-bud and mid-brain cells derived from rat embryos by a sensitive immunocytochemical technique. The cytochromes could not be detected by antibody staining at the start of the culture nor in 13-day-old embryos from which cultures were prepared. Two different antibodies directed against cytochrome P-450 revealed the ontogenic profile of the phenobarbitone-inducible b and e forms, which appeared at an earlier stage of development, day 1 of culture (equivalent to day 14 of gestation), than did the 3-methylcholanthrene-inducible c form, which appeared on day 3 of culture (equivalent to day 16 of gestation). These isoenzymes were not tissue specific. Comparison of the localization and intensity of staining of cells cultured in vitro for 5 days with tissue from the equivalent foetal developmental stage (day 18) in vivo revealed the presence of cytochrome P-450 in corresponding areas. In day 18 limb sections, cytochrome P-450 was localized in the perichondrial and myogenic tissue, which corresponded to the cells in the periphery of the chondrogenic foci in vitro. In mid-brain whole tissue, the enzyme was located in connective tissue and neurofibrils, corresponding to cells in the periphery of the foci of neurones in vitro. The correlation between in vitro and in vivo observations from time course, location and quantitative aspects, illustrated that the micromass culture technique is a valid model for metabolism studies with these specific isoenzymes.

Inducibility and functionality of rat embryonic/foetal cytochrome P-450: A study of differentiating limb-bud and mid-brain cells in vitro.

The presence of constitutive levels of cytochrome P-450 isoenzymes in cultures derived from rat embryo limb-bud (LB) and mid-brain (CNS) cells was demonstrated immunocytochemically by staining with specific monoclonal and polyclonal antibodies of cytochrome P-450. The b and e forms of cytochrome P-450 were found to be non-inducible by either in vitro co-incubation for 5 days or by transplacental maternal induction with phenobarbitone (PB), 3-methylcholanthrene (3MC) or beta-naphthoflavone (betaNF) in either cell type. Consistent with this lack of response was the observation that both in vitro and in vivo inducer treatment did not alter the toxicity of the teratogens diphenylhydantoin (DPH) or cyclophosphamide (CPA). In contrast, 3MC induction was achieved by both in vitro and transplacental regimens as gauged by the increased intensity of peroxidase staining using a monoclonal antibody to cytochrome t-450 c, in both cell types. There was also a concomitant increase in DPH toxicity (>20% gauged by a decrease in IC(50) values) in LB cells by both induction regimens but the CNS cells were refractory. betaNF induction of cytochrome P-450 was observed following in vitro and in vivo exposures in both cell types. There was no modulation of DPH or CPA toxicity after in vitro exposure to the inducers, but in vivo induction caused a strong staining reaction in both cell types, commensurate with a 30% increase in DPH toxicity in LB cells and activation of the pro-teratogen CPA. The b and e forms of cytochrome P-450 were non-inducible but it is highly likely that the c form was both inducible (by 3MC and betaNF) and functional, the latter being assessed by modulation of DPH toxicity and CPA activation. It may be possible to induce cytochrome P-450 in cells derived from embryos. The system used may be suitable for detailed investigations of the types of metabolizing systems involved in the mechanisms underlying toxicity/teratogenicity.

In vitro metabolism of teratogens by differentiating rat embryo cells.

Rapid and accurate prediction of teratogenic hazard had been achieved using cultures of differentiating limb mesenchyme (LB) and midbrain (CNS) cells from 13-day-old rat embryos. In this study we have used these cultures to examine the role of metabolism in the in vitro teratogenic activity of diphenylhydantoin (DPH) and cyclophosphamide (CPA). Two approaches were used. The first involved modulation of cytochrome P-450 activity by co-incubation in vitro with a variety of inhibitors at concentrations that were non-cytotoxic to the cells. This enhanced the toxicity of DPH by 13-82% in LB and by 3-52% in CNS cells. Benzimidazole and ellipticine caused the greatest enhancement and SKF 525A the least. DPH appears to be the proximate teratogen and there appear to be embryo-tissue cytochrome P-450s that assist in its detoxification. Following prior transplacental induction, CPA was toxic in vitro to LB cells from beta-naphthoflavone-pretreated mothers. CPA was non-toxic in cells of control, phenobarbitone- or 3-methylcholanthrene-treated embryos. Thus there appear to be inducible levels of cytochrome P-448 in embryo cells. In the second approach, positive immunocytochemical staining of the cells with both monoclonal and polyclonal P-450 antibodies identified phenobarbitone, beta-naphthoflavone- and 3-methylcholanthrene-inducible cytochrome P-450s at a constitutive level. Cytochromes P-448 (beta-naphthoflavone type) and P-450 (phenobarbitone type, PB3 fraction) were inducible, confirming that cytochrome P-450s are in fact present in the embryo cells.

A TLC fluorescence derivatization assay for chlorpromazine and its non-conjugated hepatic microsomal metabolites.

Procedures for the assay of chlorpromazine (CPZ) and its metabolites were compared. An improved assay, involving organic extraction, thin-layer chromatography and fluorescence derivatization, was developed. The compounds were extracted from microsomal protein suspensions into 15% n-propanol in dichloromethane by successive extractions at pH 2 and pH 12. CPZ and its mono- and di-desmethyl, 7-hydroxy, N-oxide, sulphoxide and N-oxide-sulphoxide metabolites were separated using TLC on silica gel developed with methanol-acetone-ammonia (50:50:1 v/v/v). The compounds were extracted from the TLC plate with chloroform-methanol (2:1 v/v) and subjected to oxidation with hydrogen peroxide. The highly fluorescent oxidized derivatives were identified as sulphoxides by their fluorescence characteristics. Amounts of derivatized CPZ and metabolites were measured using derivatized promazine as the internal standard.