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Expectations are high that the use of proteomics, gene arrays and metabonomics will improve risk assessment and enable prediction of toxicity early in drug development. These molecular profiling techniques may be used to classify compounds and to identify predictive markers that can be used to screen large numbers of chemicals. One of the challenges for the scientific community is to discriminate between changes in gene/protein expression and metabolic profiles reflecting physiological/adaptive responses, and changes related to pathology and toxicology. In these proceedings we provide a brief overview of the technologies with focus on proteomics and the possible applications to mechanistic and predictive toxicology. The discussion also includes strengths and limitations of molecular profiling technologies. [Copyright &y& Elsevier]

The metabolic activation of the promutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyI-6-phenylimidazo[4,5-b]pyridine (PhIP) by rat and mouse lung microsomes was studied using mutagenicity (strain TA98). Lungs from uninduced animals were found to activate all three compounds. A 4–6 fold higher mutagenic activity was obtained with IQ compared to MeIQx and the mutagenic response of PhIP was 1–2 orders of magnitude lower than that of IQ. In order to characterize the forms of P450 in the lung responsible for the metabolic activation of these food mutagens Western blots were performed with microsomes and partially purified P450 fractions from the lung. Western blots revealed the presence of cytochrome P450 2A, 2B and 4A forms in untreated rats. In the lung CYP 1A1 was only detectable after BNF treatment of rats. The CYP 4A isozymes, which have not previously been described in the rat lung, were further identified after PCR amplification from lung mRNA as 4A2 and 4A8. Antibody inhibition studies showed that CYP 2A3 catalyzed a major part (70%) of the metabolic activation of IQ by uninduced rat lung microsomes. The metabolic activation of MeIQx was not influenced by this antibody. An antibody against CYP 2B isozymes also partially inhibited the activation of IQ by uninduced rat lung microsomes. However, since induction of CYP 2B isozymes in the liver by phenobarbital treatment did not increase the metabolic activation of the heterocyclic amines over controls it is unlikely that the rat lung CYP 2B1 is participating in the activation of heterocyclic amines. The inhibition of the IQ-dependent mutagenicity by the CYP 2B antibody is probably due to cross-reaction with CYP 2A3. Alfa-naptho-flavone (ANF), considered to be a specific inhibitor of CYP 1A isozymes at 10 μM, partly inhibited the activation of IQ (30–40%) and MeIQx (60–80%) by uninduced rat and mouse lung microsomes. Upon pretreatment of rats with BNF, lung microsomes activated MeIQx at a rate that was 2–10-fold higher than control lung microsomes, whereas the increase in EROD activity was approximately 100-fold in the same lung preparations. These results suggest that CYP 1A1 may not be the enzyme responsible for the activation of MeIQx in the control rat despite the inhibition with ANF. It is likely that ANF can inhibit other P450 enzymes in the lung, including CYP 2A3. The involvement of CYP 2A3. The involvement of CYP 2A3 in the metabolic activation of IQ by uninduced rat lung shows that CYP forms that are not of major importance in the liver may play a significant role in extra-hepatic activation of heterocyclic amines. [ABSTRACT FROM PUBLISHER]

Abstract: Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Two forms of ALT have been identified, ALT1 and ALT2, encoded by separate genes. The cellular and tissue distribution of the different ALT proteins has not been characterized in humans, and their relative contribution to serum is unknown. Here, we describe the development of novel isoenzyme specific ALT1 and ALT2 antibodies and the expression of the enzymes in human cells and organs. In normal human tissue, high expression of ALT1 was found in liver, skeletal muscle and kidney and low levels in heart muscle and not detectable in pancreas. High ALT2 reactivity was detected in heart and skeletal muscle, while no ALT2 expression was found in liver or kidney. Using immunohistochemistry, strong ALT1 reactivity was found in hepatocytes, renaltubular epithelial cells and in salivary gland epithelial cells, while ALT2 was expressed in adrenal gland cortex, neuronal cell bodies, cardiac myocytes, skeletal muscle fibers and endocrine pancreas. Immunoprecipitation using ALT antibodies on normal human serums showed ALT1 to be mainly responsible for basal ALT activity. Together, the results points to a differential expression of ALT1 and ALT2 in human organs and substantiate a need for investigations regarding the possible impacts on ALT measurements. [Copyright &y& Elsevier]

High-throughput biomolecular profiling techniques such as transcriptomics, proteomics and metabolomics are increasingly being used in in vivo studies to recognize and characterize effects of xenobiotics on organs and systems. Of particular interest are biomarkers of treatment-related effects which are detectable in easily accessible biological fluids such as blood. A fundamental challenge in such biomarker studies is selecting among the plethora of biomolecular changes induced by a compound and revealed by molecular profiling, to identify biomarkers which are exclusively or predominantly due to specific processes. In this work we present a cross-compartment correlation network approach, involving no a priori supervision or design, to integrate proteomic, metabolomic and transcriptomic data for selecting circulating biomarkers. The case study we present is the identification of biomarkers of drug-induced hepatic toxicity effects in a rodent model. Biomolecular profiling of both blood plasma and liver tissue from Wistar Hannover rats administered a toxic compound yielded many hundreds of statistically significant molecular changes. We exploited drug-induced correlations between blood plasma analytes and liver tissue molecules across study animals in order to nominate selected plasma molecules as biomarkers of drug-induced hepatic alterations of lipid metabolism and urea cycle processes.