Proteomic profiling of rectal cancer reveals acid ceramidase is implicated in radiation response.

BACKGROUND: Neoadjuvant chemoradiotherapy (CRT) is used in locally advanced rectal cancer when tumours threaten the circumferential resection margin, with varying response to treatment. This experimental study aimed to identify significantly differentially expressed proteins between patients responding and not responding to CRT, and to validate any proteins of interest. METHODS: Mass spectrometry (with isobaric tagging for relative quantification) analysis of rectal cancers pre- and post-CRT, and at resection. Validation of proteins of interest was performed by assessing tissue microarray (TMA) immunohistochemistry expression in a further 111 patients with rectal cancer. RESULTS: Proteomic data are available via ProteomeXchange with identifier PXD008436. Reduced abundance of contributing peptide ions for acid ceramidase (AC) (log fold change -1.526, p = 1.17E-02) was observed in CRT responders. Differential expression of AC was confirmed upon analysis of the TMAs. Cancer site expression of AC in stromal cells from post-CRT resection specimens was observed to be relatively low in pathological complete response (p = 0.003), and relatively high with no response to CRT (p = 0.017). CONCLUSION: AC may be implicated in the response of rectal cancer to CRT. We propose its further assessment as a novel potential biomarker and therapeutic target. SIGNIFICANCE: There is a need for biomarkers to guide the use of chemoradiotherapy in rectal cancer, as none are in routine clinical use. We have determined acid ceramidase may have a role in radiation response, based on novel proteomic profiling and validation in a wider dataset using tissue microarrays. The ability to predict or improve response would positively select those patients who will derive benefit, prevent delays in the local and systemic management of disease in non-responders, and reduce morbidity associated with chemoradiotherapy.

Exome sequencing of synchronously resected primary colorectal tumours and colorectal liver metastases to inform oncosurgical management.

BACKGROUND: Next generation sequencing technology has facilitated mapping of the colorectal cancer genotype and furthered our understanding of metastogenesis. The aim of this study was to investigate for conserved and different mutations in the exomes of synchronously resected primary colorectal tumour and liver metastases. This information could potentially be utilised to guide the treatment of advanced disease with the help of biological information from the primary tumour. METHODS: We performed exome sequencing of synchronously resected primary colorectal cancer and colorectal liver metastases as well as normal colonic mucosa and liver parenchyma, from four patients who had received neo-adjuvant chemotherapy, at a depth of 50X using the Ion Proton platform. Raw data was mapped to the reference genome prior to variant calling, annotation and downstream analysis. RESULTS: Exome sequencing identified 585 non-synonymous missense single nucleotide variants (SNVs), of which 215 (36.8%) were unique to the primary tumour, 226 (38.6%) unique to the metastasis and 81 (13.8%) present in patient matched pairs. SNVs identified in the ErbB pathway appear to be concordant between primary and metastatic tumours. CONCLUSION: Only 13.8% of the metastatic exome can be predicted by the genotype of the primary tumour. We have demonstrated concordance of a number of SNVs in the ErbB pathway, which may inform selection of therapeutic agents in advanced colorectal cancer.

Immunohistochemical hENT1 expression as a prognostic biomarker in patients with resected pancreatic ductal adenocarcinoma undergoing adjuvant gemcitabine-based chemotherapy.

BACKGROUND: Human equilibrative nucleoside transporters (hENTs) are transmembranous proteins that facilitate the uptake of nucleosides and nucleoside analogues, such as gemcitabine, into the cell. The abundance of hENT1 transporters in resected pancreatic ductal adenocarcinoma (PDAC) might make hENT1 a potential biomarker of response to adjuvant chemotherapy. The aim of this study was to see whether hENT1 expression, as determined by immunohistochemistry, was a suitable predictive marker for subsequent treatment with gemcitabine-based adjuvant chemotherapy. METHODS: A systematic review was performed, searching databases from January 1997 to January 2016. Articles pertaining to hENT1 immunohistochemical analysis in resected PDAC specimens from patients who subsequently underwent adjuvant gemcitabine-based chemotherapy were identified. Eligible studies were required to contain survival data, reporting specifically overall survival (OS) and disease-free survival (DFS) with associated hazard ratios (HRs) stratified by hENT1 status. RESULTS: Of 42 articles reviewed, eight were suitable for review, with seven selected for quantitative meta-analysis. The total number of patients included in the meta-analysis was 770 (405 hENT1-negative, 365 hENT1-positive). Immunohistochemically detected hENT1 expression was significantly associated with both prolonged DFS (HR 0·58, 95 per cent c.i. 0·42 to 0·79) and OS (HR 0·52, 0·38 to 0·72) in patients receiving adjuvant gemcitabine but not those having fluoropyrimidine-based adjuvant therapy. CONCLUSION: Expression of hENT1 is a suitable prognostic biomarker in patients undergoing adjuvant gemcitabine-based chemotherapy.

Evaluation of a novel tissue stabilization gel to facilitate clinical sampling for translational research in surgical trials.

BACKGROUND: The aim was to establish the feasibility of using a tissue stabilization gel (Allprotect™) as an alternative to liquid nitrogen to facilitate collection of clinical samples for translational research. METHODS: Tumour samples from patients undergoing surgery for primary or metastatic colorectal cancer were either snap-frozen in liquid nitrogen or stored in Allprotect™ under a number of different conditions. Sample integrity was compared across different storage conditions by assessing biomolecule stability and function. DNA quality was assessed spectrophotometrically and by KRas genotyping by pyrosequencing. Total RNA retrieval was determined by nanodrop indices/RNA integrity numbers, and quality assessed by reverse transcription-PCR for two representative genes (high-mobility group box 1, HMGB1; carboxylesterase 1, CES1) and two microRNAs (miR122 and let7d). Western blot analysis of HMGB1 and CES1 was used to confirm protein expression, and the metabolic conversion of irinotecan to its active metabolite, SN-38, was used to assess function. RESULTS: Under short-term storage conditions (up to 1 week) there was no apparent difference in quality between samples stored in Allprotect™ and those snap-frozen in liquid nitrogen. Some RNA degradation became apparent in tissue archived in Allprotect™ after 1 week, and protein degradation after 2 weeks. CONCLUSION: In hospitals that do not have access to liquid nitrogen and -80°C freezers, Allprotect™ provides a suitable alternative for the acquisition and stabilization of clinical samples. Storage proved satisfactory for up to 1 week, allowing transfer of samples without the need for specialized facilities. Surgical relevance Access to clinical material is a fundamental component of translational research that requires significant infrastructure (research personnel, liquid nitrogen, specialized storage facilities). The aim was to evaluate a new-to-market tissue stabilization gel (Allprotect™), which offers a simple solution to tissue preservation without the need for complex infrastructure. Allprotect™ offers comparable DNA, RNA and protein stabilization to tissue snap-frozen in liquid nitrogen for up to 1 week. Degradation of biomolecules beyond this highlights its role as a short-term tissue preservative. Allprotect™ has the potential to increase surgeon participation in translational research and surgical trials requiring tissue collection.

Hepatic activation of irinotecan predicts tumour response in patients with colorectal liver metastases treated with DEBIRI: exploratory findings from a phase II study.

PURPOSE: The response of colorectal liver metastases to the cytotoxic agent irinotecan varies widely. Attempts to correlate tumour metabolism with response have been mixed. This study investigated the hepatic metabolism of irinotecan as a potential predictor of tumour response to irinotecan-eluting beads (DEBIRI). METHODS: Ten patients with colorectal liver metastases were treated with 200 mg irinotecan (as DEBIRI) as part of the PARAGON II study. Hepatic expression of key metabolising enzymes was measured using mass spectrometry-based proteomics. Serum drug concentrations and hepatic irinotecan metabolism were characterised and correlated with tumour response. RESULTS: Serum concentrations of irinotecan metabolites did not correlate with hepatic metabolism or pathological response. There was a strong correlation between hepatic CES-2 expression and activation of irinotecan (r (2) = 0.96, p < 0.001). Patients with a UGT1A1*28 6/7 SNP showed no difference in drug metabolism or pathological response. Hepatic CES-2 mediated activation of irinotecan clearly correlated with tumour replacement by fibrosis (r (2) = 0.54, p = 0.01). CONCLUSION: This study provides the first evidence that hepatic activation of irinotecan predicts tumour response. Delivery of liver-targeted irinotecan to normal liver tissue rather than tumour may be a more rational approach to maximise response.

iTRAQ reveals candidate pancreatic cancer serum biomarkers: influence of obstructive jaundice on their performance.

BACKGROUND: The aims of our study were to identify serum biomarkers that distinguish pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) patients from benign pancreatic disease patients and healthy subjects, and to assess the effects of jaundice on biomarker performance. METHODS: Isobaric tags for relative and absolute quantification were used to compare pooled serum and pancreatic juice samples from a test set of 59 and 25 subjects, respectively. Validation was undertaken in 113 independent subjects. RESULTS: Candidate proteins Complement C5, inter-α-trypsin inhibitor heavy chain H3, α1-ß glycoprotein and polymeric immunoglobulin receptor were elevated in cancer, as were the reference markers CA19-9 and Reg3A. Biliary obstruction had a significant effect on the performance of the markers, in particular within the PDAC group where the presence of jaundice was associated with a significant increase in the levels of all six proteins (P<0.01). Consequently, in the absence of jaundice, proteins showed reduced sensitivity for PDAC patients over benign subjects and healthy controls (HCs). Similarly, in the presence of jaundice, markers showed reduced specificity for PDAC patients over benign subjects with jaundice. Combining markers enabled improved sensitivity for non-jaundiced PDAC patients over HCs and improved specificity for jaundiced PDAC patients over jaundiced benign disease subjects. CONCLUSIONS: The presence-absence of jaundice in the clinical scenario severely impacts the performance of biomarkers for PDAC diagnosis and has implications for their clinical translation.

S100A6 binds to annexin 2 in pancreatic cancer cells and promotes pancreatic cancer cell motility.

BACKGROUND: High levels of S100A6 have been associated with poor outcome in pancreatic cancer patients. The functional role of S100A6 is, however, poorly understood. METHODS: Immunoprecipitation followed by two-dimensional gel electrophoresis and mass spectrometry were undertaken to identify S100A6 interacting proteins in pancreatic cancer cells. Immunohistochemistry and coimmunofluorescence were performed to examine expression or colocalisation of proteins. siRNA was used to deplete specific proteins and effects on motility were measured using Boyden Chamber and wound healing assays. RESULTS: Our proteomic screen to identify S100A6 interacting proteins revealed annexin 11, annexin 2, tropomyosin beta and a candidate novel interactor lamin B1. Of these, annexin 2 was considered particularly interesting, as, like S100A6, it is expressed early in the development of pancreatic cancer and overexpression occurs with high frequency in invasive cancer. Reciprocal immunoprecipitation confirmed the interaction between annexin 2 and S100A6 and the proteins colocalised, particularly in the plasma membrane of cultured pancreatic cancer cells and primary pancreatic tumour tissue. Analysis of primary pancreatic cancer specimens (n=55) revealed a strong association between high levels of cytoplasmic S100A6 and the presence of annexin 2 in the plasma membrane of cancer cells (P=0.009). Depletion of S100A6 was accompanied by diminished levels of membrane annexin 2 and caused a pronounced reduction in the motility of pancreatic cancer cells. CONCLUSION: These findings point towards a functional role for S100A6 that may help explain the link between S100A6 expression in pancreatic cancer and aggressive disease.

alpha(1)-Adrenoceptor antagonists prevent paracetamol-induced hepatotoxicity in mice.

BACKGROUND AND PURPOSE: Paracetamol, a major cause of acute liver failure (ALF) represents a significant clinical problem. Adrenoceptor stimulation or antagonism can modulate chemical-induced hepatotoxicity. We investigated the role of endogenous catecholamines and alpha(1)-adrenoceptors in the development of paracetamol- induced hepatotoxicity. EXPERIMENTAL APPROACH: Paracetamol (3.5 mmol kg(-1)) was administered to male CD-1 mice, with and without alpha(1)-adrenoceptor antagonists (prazosin, doxazosin, terazosin and tamsulosin; 35.7 micromol kg(-1)). Serum transaminases and hepatic glutathione (GSH) levels were assessed as markers of hepatic damage. Paracetamol bioactivation was assessed by covalent binding, hepatic and urinary conjugate formation and uridine glucuronosyltransferase activity. Plasma catecholamines levels and hepatic congestion were also analysed. KEY RESULTS: Plasma catecholamine levels were significantly elevated 5 h post paracetamol administration. Prazosin prevented hepatotoxicity when administered 1 h before a toxic paracetamol insult and importantly, when administered up to 1 h post paracetamol injection. Prazosin had no effect on paracetamol-induced depletion of hepatic GSH, paracetamol bioactivation or paracetamol-induced transcription of defence genes. Paracetamol toxicity is associated with marked accumulation of erythrocytes within hepatic sinusoids and prazosin completely prevented this accumulation. CONCLUSION AND IMPLICATIONS: Paracetamol-induced hepatocellular damage is associated with increased circulating catecholamines. alpha(1)-Adrenoceptor antagonists conferred complete protection from paracetamol -induced hepatotoxicity. Protection was associated with absence of hepatic erythrocyte accumulation. Increased catecholamine levels may contribute to the pathophysiology of paracetamol-induced hepatotoxicity by compromising hepatic perfusion. Protection against paracetamol toxicity by alpha(1) antagonists in mice has implications for therapeutic management of patients presenting with paracetamol overdose and ALF.

Are chemically reactive metabolites responsible for adverse reactions to drugs?

Low molecular weight organic chemicals can be transformed by normal drug-metabolising systems into short-lived metabolites that are inherently reactive towards cellular macromolecules. There is direct evidence that the formation of such chemically reactive metabolites may lead to mutagenesis, carcinogenicity, apoptosis and necrosis in both cell and animal models. A number of drugs associated with non-pharmacological drug toxicities in man have been shown to undergo bioactivation either in vivo or in vitro. We have therefore examined the evidence for the role of reactive metabolites in the three most common drug-induced toxicities: hepatotoxicity, skin reactions and blood dyscrasias.

Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi.

Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This toxic reaction is associated with metabolic activation by the P450 system to form a quinoneimine metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione, a reaction catalyzed in part by the glutathione S-transferases (GST), such as GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we examined acetaminophen metabolism and liver damage in mice nulled for GstP (GstP1/P2((-/-))). Contrary to our expectations, instead of being more sensitive, GstP null mice were highly resistant to the hepatotoxic effects of this compound. No significant differences between wild-type (GstP1/P2((+/+))) mice and GstP1/P2((-/-)) nulls in either the rate or route of metabolism, particularly to glutathione conjugates, or in the levels of covalent binding of acetaminophen-reactive metabolites to cellular protein were observed. However, although a similar rapid depletion of hepatic reduced glutathione (GSH) was found in both GstP1/P2((+/+)) and GstP1/P2((-/-)) mice, GSH levels only recovered in the GstP1/P2((-/-)) mice. These data demonstrate that GstP does not contribute in vivo to the formation of glutathione conjugates of acetaminophen but plays a novel and unexpected role in the toxicity of this compound. This study identifies new ways in which GST can modulate cellular sensitivity to toxic effects and suggests that the level of GST Pi may be an important and contributing factor in the sensitivity of patients with acetaminophen-induced hepatotoxicity.

Hepatocellular response to chemical stress in CD-1 mice: induction of early genes and gamma-glutamylcysteine synthetase.

Exposure of cells to toxic chemical species can result in reduced glutathione (GSH) depletion, generation of free radicals, and/or binding to critical cell determinants. Chemical stress is usually followed by a concerted cellular response aimed at restoring homeostasis, although the precise initial stimulus for the response is unclear. We have focused on one component of this stress response, the up-regulation of gamma-glutamylcysteine synthetase (gamma-GCS) and the preceding molecular events involved in its regulation in an in vivo mouse model. Male CD-1 mice received buthionine sulphoximine (BSO; 7.2 mmol/kg), diethyl maleate (DEM; 4.2 mmol/kg), paracetamol (APAP; 3.5 and 1.0 mmol/kg), or carbon tetrachloride (CCl(4); 1.0 and 0.2 mmol/kg). Biochemical (serum transaminase and hepatic GSH levels) and molecular (c-jun and c-fos messenger RNA [mRNA] levels and activator protein 1 [AP-1] DNA binding activity) parameters were measured, as well as the consequent effects on gamma-GCS levels and activity. All compounds produced GSH depletion, but only the higher doses of APAP and CCl(4) caused liver damage. DEM, APAP, and CCl(4) increased c-jun and c-fos mRNA levels, together with an increase in AP-1 binding; BSO failed to induce AP-1 despite an increase in c-fos. Interestingly, the effects on gamma-GCS varied markedly according to the compound: BSO and DEM increased gamma-GCS enzyme activity, although only DEM, but not BSO, resulted in an increase in gamma-GCS(h) mRNA and protein. In contrast, APAP and CCl(4) both increased gamma-GCS(h) mRNA and protein; however, there was a marked dose-dependent decrease in gamma-GCS activity. These data indicate that the effect of chemical stress on the liver is compound specific and is not merely dependent on depletion of GSH.

Cellular disposition of sulphamethoxazole and its metabolites: implications for hypersensitivity.

1. Bioactivation of sulphamethoxazole (SMX) to chemically-reactive metabolites and subsequent protein conjugation is thought to be involved in SMX hypersensitivity. We have therefore examined the cellular metabolism, disposition and conjugation of SMX and its metabolites in vitro. 2. Flow cytometry revealed binding of N-hydroxy (SMX-NHOH) and nitroso (SMX-NO) metabolites of SMX, but not of SMX itself, to the surface of viable white blood cells. Cellular haptenation by SMX-NO was reduced by exogenous glutathione (GSH). 3. SMX-NHOH and SMX-NO were rapidly reduced back to the parent compound by cysteine (CYS), GSH, human peripheral blood cells and plasma, suggesting that this is an important and ubiquitous bioinactivation mechanism. 4. Fluorescence HPLC showed that SMX-NHOH and SMX-NO depleted CYS and GSH in buffer, and to a lesser extent, in cells and plasma. 5. Neutrophil apoptosis and inhibition of neutrophil function were induced at lower concentrations of SMX-NHOH and SMX-NO than those inducing loss of membrane viability, with SMX having no effect. Lymphocytes were significantly (P<0.05) more sensitive to the direct cytotoxic effects of SMX-NO than neutrophils. 6. Partitioning of SMX-NHOH into red blood cells was significantly (P<0.05) lower than with the hydroxylamine of dapsone. 7. Our results suggest that the balance between oxidation of SMX to its toxic metabolites and their reduction is an important protective cellular mechanism. If an imbalance exists, haptenation of the toxic metabolites to bodily proteins including the surface of viable cells can occur, and may result in drug hypersensitivity.

Expression of cytochrome P4502E1 in human liver: assessment by mRNA, genotype and phenotype.

Cytochrome P4502E1 (CYP2E1) is constitutively expressed in human liver and is responsible for the metabolic bioactivation of a wide variety of xenobiotics, including a number of protoxins and procarcinogens. CYP2E1 expression is regulated at several levels including pre-transcriptional, transcriptional and post-transcriptional levels, and any variation in enzyme concentration and hence activity may represent increased risk of toxicity or carcinogenicity. We have investigated variability in the levels of CYP2E1 mRNA, protein and functional activity in a human liver bank, and attempted to relate these parameters to the RsaI restriction fragment length polymorphism in the 5'-flanking region. Variation in CYP2E1 mRNA (18-fold) was greater than the variation seen in CYP2E1 protein (twofold) and functional activity (fourfold) determined using two probe substrates, chlorzoxazone and p-nitrophenol. Although protein and functional activity showed a significant correlation (r = 0.93 and r = 0.83 for chlorzoxazone and p-nitrophenol, respectively), there was no correlation between any of these parameters and mRNA levels. Also, the variation in CYP2E1 activity could not be directly accounted for by the RsaI polymorphism in our samples. In conclusion, our results are consistent with a complex regulation of CYP2E1 and the fact that it is highly conserved in the human population. The absence of a relationship between the RsaI polymorphism and CYP2E1 activity is consistent with other studies performed in Caucasians, but does not exclude an effect of this polymorphism on inducibility of CYP2E1.

Relevance of induction of human drug-metabolizing enzymes: pharmacological and toxicological implications.

1. Human drug-metabolizing systems can be induced, or activated, by a large number of exogenous agents including drugs, alcohol, components in the diet and cigarette smoke, as well as by endogenous factors. 2. Such perturbation of enzyme activity undoubtedly contributes to both intra-and inter-individual variation both with respect to the rate and route of metabolism for a particular drug. Induction may, in theory, either attenuate the pharmacological response or exacerbate the toxicity of a particular drug, or both. 3. The clinical impact of enzyme induction will depend upon the number of different enzyme isoforms affected and the magnitude of the inductive response within an individual, and also on the therapeutic indices of the affected substrates. 4. The toxicological implications will be determined either by any change in the route of metabolism, or by a disturbance of the balance between activation and detoxication processes, which may be isozyme selective.

Detection of autoantibodies directed against human hepatic endoplasmic reticulum in sera from patients with halothane-associated hepatitis.

1. Previous studies have demonstrated the presence of antibodies to trifluoroacetylated hepatic proteins (TFA-proteins) in sera from patients with the severe form of halothane-associated hepatitis (halothane hepatitis). The TFA-proteins are produced via cytochrome P450-mediated metabolism of halothane to the reactive species TFA-chloride. 2. To investigate the presence of autoantibodies (which recognize various non-TFA-modified human hepatic polypeptides) in patients with halothane hepatitis immunoblotting experiments were performed using microsomal fractions prepared freshly from livers of five different (halothane-free) tissue donors. Blots were developed using 15 well-characterised sera from patients with halothane hepatitis. 3. Autoantibodies to human hepatic polypeptides were detected in most, but not all, of the patients' sera. The pattern of antibody reactivity varied markedly between sera. Although no common pattern of antibody recognition was observed, polypeptides of molecular mass between 60 and 80 kDa were the predominant targets. A similar protein recognition pattern was seen when each positive serum was tested against the five individual human liver samples. 4. Such autoantibodies were not detected in sera from 16 normal human blood donors, but were detected in three of six sera from patients exposed to halothane without developing hepatitis. 5. The autoantibodies are thought to arise in patients exposed to halothane as a consequence of a halothane-induced immune response to chemically-modified proteins. Such antibodies could contribute to the complex pathological processes involved in halothane hepatitis.

The role of cytochrome P450 enzymes in hepatic and extrahepatic human drug toxicity.

The human cytochrome P450 enzyme system metabolises a wide array of xenobiotics to pharmacologically inactive metabolites, and occasionally, to toxicologically active metabolites. Impairment of cytochrome P450 activity, which may be either genetic or environmental, may lead to toxicity caused by the parent compound itself. In practise, this usually only applies to drugs that have a narrow therapeutic index and when their clearance is critically dependent upon the fraction normally metabolised by that pathway. P450 enzymes may also convert the drug to a chemically reactive metabolite, which, if not detoxified, may lead to various forms of hepatic and extrahepatic toxicity, including cellular necrosis, hypersensitivity, teratogenicity, and carcinogenicity, depending on the site of formation and the relative stability of the metabolite, and the cellular macromolecule with which it reacts. Variation in the regulation and expression of the drug metabolising enzymes may play a key role in both interindividual variation in sensitivity to drug toxicity and tissue-specific damage. Avoidance of toxicity may be possible in rare instances by prediction of individual susceptibility or by designing new chemical entities that are metabolised by a range of enzymes (both cytochromes P450 and others) and do not undergo bioactivation.

Inhibition of caffeine metabolism by ciprofloxacin in children with cystic fibrosis as measured by the caffeine breath test.

The caffeine breath test was carried out in six children with cystic fibrosis, before and during a course of ciprofloxacin. There was a significant decrease in the 2 h cumulative labelled CO2 exhaled during ciprofloxacin treatment, mean difference (s.d.) -5.2(3.3)%, P < 0.02. The results suggest an inhibition of 3-N-demethylation of caffeine (CYP1A2 enzyme activity) by ciprofloxacin. Ciprofloxacin may cause significant drug interactions in children with cystic fibrosis. The caffeine breath test can be used to study drug interactions involving CYP1A2 in children.

An investigation of the formation of cytotoxic, genotoxic, protein-reactive and stable metabolites from naphthalene by human liver microsomes.

Chemically reactive epoxide metabolites have been implicated in various forms of drug and chemical toxicity. Naphthalene, which is metabolized to a 1,2-epoxide, has been used as a model compound in this study in order to investigate the effects of perturbation of detoxication mechanisms on the in vitro toxicity of epoxides in the presence of human liver microsomes. Naphthalene (100 microM) was metabolized to cytotoxic, protein-reactive and stable, but not genotoxic, metabolites by human liver microsomes. The metabolism-dependent cytotoxicity and covalent binding to protein of naphthalene were significantly higher in the presence of phenobarbitone-induced mouse liver microsomes than with human liver microsomes. The ratio of trans-1,2-dihydrodiol to 1-naphthol was 8.6 and 0.4 with the human and the induced mouse microsomes, respectively. The metabolism-dependent toxicity of naphthalene toward human peripheral mononuclear leucocytes was not affected by the glutathione transferase mu status of the co-incubated cells. Trichloropropene oxide (TCPO; 30 microM), an epoxide hydrolase inhibitor, increased the human liver microsomal-dependent cytotoxicity (19.6 +/- 0.9% vs 28.7 +/- 1.0%; P = 0.02) and covalent binding to protein (1.4 +/- 0.3% vs 2.8 +/- 0.2%; P = 0.03) of naphthalene (100 microM), and reversed the 1,2-dihydrodiol to 1-naphthol ratio from 6.6 (without TCPO) to 2.6, 0.6 and 0.1 at TCPO concentrations of 30, 100 and 500 microM, respectively. Increasing the human liver microsomal protein concentration reduced the cytotoxicity of naphthalene, while increasing its covalent binding to protein and the formation of the 1,2-dihydrodiol metabolite. Co-incubation with glutathione (5 mM) reduced the cytotoxicity and covalent binding to protein of naphthalene by 68 and 64%, respectively. Covalent binding to protein was also inhibited by gestodene, while stable metabolite formation was reduced by gestodene (250 microM) and enoxacin (250 microM). The study demonstrates that human liver cytochrome P450 enzymes metabolize naphthalene to a cytotoxic and protein-reactive, but not genotoxic, metabolite which is probably an epoxide. This is rapidly detoxified by microsomal epoxide hydrolase, the efficiency of which can be readily determined by measurement of the ratio of the stable metabolites, naphthalene 1,2-dihydrodiol and 1-naphthol.

The mechanism of bioactivation and antigen formation of amodiaquine in the rat.

A glutathione conjugate of amodiaquine has been isolated and characterized from rat bile after administration of [14C]amodiaquine (50 mumol/kg, 5.0 muCi/rat) to anaesthetized male Wistar rats. Thioether conjugates of amodiaquine in rat bile accounted for a total of 12% of the dose, 5 hr after administration of the drug. In addition, 1% of the dose remained in the liver covalently bound to tissue proteins after 5 hr. These findings provide direct evidence that a chemically reactive metabolite, amodiaquine quinoneimine, has been formed from the drug in vivo. A second major metabolite, desethylamodiaquine, accounting for 14% of the given dose, was present in the liver after 5 hr. Enzyme inhibition studies with ketoconazole-pretreated rats showed that both amodiaquine quinoneimine and desethylamodiaquine formation can be catalysed by cytochrome P450. The demonstration that amodiaquine readily and extensively forms a metabolite in vivo, with strong reactivity towards protein and non-protein thiol groups, may help to explain the idiosyncratic toxicity observed in man.

An investigation of the formation of cytotoxic, protein-reactive and stable metabolites from carbamazepine in vitro.

The formation of chemically reactive metabolites from carbamazepine (CBZ) in the presence of mouse and human liver microsomes has been investigated using cytotoxicity and irreversible binding of radiolabelled compound as quantitative end-points. For comparison, the formation of the stable CBZ-10,11-epoxide (CBZ-10,11-E) has been measured. The formation of the cytotoxic, protein-reactive and stable metabolites of CBZ was increased by induction of the cytochrome P450 enzymes by phenobarbitone and reduced by co-incubation in vitro with ketoconazole (10-250 microM), suggesting that the formation of these metabolites is cytochrome P450 dependent. All human livers tested (N = 6) bioactivated CBZ to a protein-reactive metabolite, the mean covalent binding increasing from 0.08 +/- 0.01% (without NADPH) to 0.27 +/- 0.09% (with NADPH; P less than or equal to 0.05). The formation of the chemically reactive metabolites was reduced by a subphysiological concentration of reduced glutathione (GSH) (500 microM), while ascorbic acid (100 microM) had no effect. Neither compound affected the formation of CBZ-10,11-E. Microsomal epoxide hydrolase (mEH), but not cytosolic epoxide hydrolase, caused a concentration-dependent inhibition of cytotoxicity reaching a maximum of 60% at 100 U of mEH. Covalent binding was also reduced by 60% by 100 U mEH. The separated T- and B-lymphocytes showed no difference in sensitivity when incubated with CBZ and mouse microsomes. The study demonstrates that the balance between activation of CBZ by the cytochrome P450 enzymes to a chemically reactive arene oxide metabolite and its detoxification by mEH and GSH may contribute to individual susceptibility to CBZ idiosyncratic toxicity.