Mechanistic relevance of peroxisome proliferation to human cancer risk assessment

Symp osium 3. Peroxisomal Activation: Its Physiological and Pathological Consequences

such as neurological , cardiovascular, reproductive or endocrinological dysfunction. Chemical modulation or oncogene down-regulation of GJIC in initiated tissues has been shown to lead to tumor promotion. Genetic syndromes carrying a mutated gap j unction gene, together with some transgenic and knock-out gap junction gene mice, provide evidence for the importan ce of this organelle found only in metazoans. Implications for "threshold effects" to toxicants and for risk assessment are evident. Research supported by the NCI (CA2 1004), NIEHS (P42 ES04911-08, EPR! (UBP950822) and USAFOSR (F49620-97- 1 -0022).

DOWN-REGULATION OF PROTEIN KINASE C: A POTENTIAL MECHANISM FOR 2-AMINO-3-METHYLIMIDAZO[4,5-F] QUINOLONE-MEDIATED IMMUNOSUPPRESSION

K.H. Yang *, Y.w. Lee, Y.J. Jeon. Korea Advanced Institute of Science and Technology, Taejon 305-701 , Korea 2-Amino-3-methylimidazo[4,5-flquinoline (lQ ) is one of several mutagenic heterocyclic amines formed during the cooking of proteinaceous foods. IQ is a potent mutagen in the Ames Salmonella assay, and has been found to be carcinogenic in rodents and nonhuman primates. In the present study, we investigated the mechanism of IQ-induced immunosuppression . A significant and dose-related suppression of the proliferative response to concanavalin A (Con A) or PMA and ionomycin was observed by the treatment of IQ. IQ also inhibited the production of IL-I and IL-2 which are important in the immune respon se. To investigate the mechanism of IQ-induced immunosuppression, we assessed the effect of IQ on the activation of protein kinase C (PKC). IQ decreased PKC activity in both the membrane and the cytosolic fraction and inhibited the phosphorylation of MARCKS , myristoylated alanine rich C kinase substrate. Since the activations of transcription factors NF-KBlRel and AP- l are known to regulated by PKC, the effects of IQ on the activations of these transcription factors were also studied using a transient transfection assay and electrophoretic mobility-shift assay. The activations of NF-KBlRel and AP-I were markedly inhibited by IQ in Con A-stimulated Jurkat T cells. These data suggested that the suppression of Iymphoproliferation and cytokine production by IQ might be associated with the down-regulation of PKC activity. (Grants from the Ministry of Science and Technology, Korea)

83. Peroxisomal Activation: Its Physiological and Pathological Consequences

I S3/L 1 I MECHANISM OF PPAR ACTION

specific DR-I response elements (PPRE) in the promoters of respective genes. Treatment with PP results in rodents in hypolipidaemia , peroxisome proliferation and liver hypertrophy and hyperplasia leading to nongenotoxic hepatocarcinogenesis. In contrast to rodents, the hypolipidaemic effect exerted by PP in humans is not accompanied by peroxisome proliferation nor by induction of peroxisomal p -oxidation or other activities induced by PP in rodents. Similarly, peroxisome proliferation induced by PP in cultured rat hepatocytes is not induced in human hepatoma cell lines or primary cultured human hepatocytes. Since PPARa has been cloned in humans and PPREs have been characterized in respective human promoters, nonresponsiveness in humans has been ascribed to a significant [ower liver content of hPPARa accompanied by pronounced interindividual variability, to the abundance of a spliced hPPARa variant or repression of hPPARa by hNUC I. Accounting however for nonresponsiveness still leaves unsolved the hypolipidemic mode of action of PP in humans.

IS3/L31 PPAR«-REGULATED GROWTH RESPONSES AND THEIR IMPORTANCE TO HEPATOCARCINOGENESIS

N.H. James, N.J. Woodyatt, N. Macdonald , M. Rolfe, S.C. Hasmall , P.R. Holden, J.D. Tugwood , R.A. Roberts *. Cancer Biology Group, Zen eca Central Toxicology Laboratory, Alderley Park, Macclesfield . SK10 4TJ. UK Peroxisome proliferators (PPs) are a class of nongenotoxic rodent hepatocarcinogens that act by perturbing liver growth regulation. We have demonstrated that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by transform ing growth factor-S I (TGFp I), DNA damage or ligation of Fas I , a receptor related to the tumour necrosis factor a (TNFa ) family of cell surface receptors . PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha, PPARa, a member of the nuclear hormone receptor superfamily. We investigated whether activation of PPARa mediates the suppression of rat hepatocyte apoptosis ind uced by PPs. We isolated a naturally occurring variant form of PPARa (hPPARa -6129) from human liver by PCR cloning. hPPARa -6/29 shared the ability of mPPARa to bind to DNA but, unlike mPPARa , could not be activated by PPs. Furthermore, hPPARa-6129 could act as a dominant negative regulator of PPAR-mediated gene transcriptiorr", When introduced into primary rat liver cell cultures by transient transfection, hPPARa -6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin , but not that seen in response to phenobarbitone (PB), a nongenotoxic carcinogen whose action does not involve PPAR2. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. Recent data have suggested that TNFa , perhaps released by liver Kupffer cells in response to PPs, may playa key role in mediating the effects of PPs on hepatocyte growth regulatiorr' .

Abstract not received at time of publication.

[I] Gill, J. II.• Roberts. R. A. & Dive, C. Carcinogene sis. In press (1998). [2] Roberts. R. A. et al. Carcinogenesis 19, In press ( 1998). [3] Rolfe, M. & Roberts. R. A. Carcinogenesis 18, 2277- 2280 ( 1997).

IS3/L21 PPAR« ACTIVATION AND ITS CONSEQUENCES IN

IS3/L4

J.A. Gustafson. Sweden

HUMANS

J. Bar-Tana, Department of Human N utrition and Metabolism, Hebrew University Medical School, Jerusalem 91120, Israel Native and xenobiotic amphipathic carboxylates collectively defined as peroxisome proliferators (PP) induce in rodents a pleiotropic effect consisting of activation of expression of genes coding for peroxisomal p-oxidation, cytochrome P-450-catalyzed w-oxidation, and other activities. Transcriptional activation is mediated by peroxiso me proliferators-activated receptor a (PPARa) which binds to

5

1

MECHANISTIC RELEVANCE OF PEROXISOME PROLIFERATION TO HUMAN CANCER RISK ASSESSMENT

J.K. Reddy. Northwestern University Medical School, Chicago, IL, USA Sustained induction of peroxisome proliferation leads to the development of liver tumors in rats and mice exposed to peroxisome proliferators despite their nonmutagenic nature. Two mechanistic issues are important for consideration: i) upstream events responsible for the tissue and species specific induction of the characteristic

Symp osium 4. New Trends in Forensic Tox icology

6

pleiotropic responses by peroxisome proliferators, and ii) downstream events that are responsible for the development of liver tumors in species that are sensitive to the induction of peroxisome proliferation. Tissue and species responses to peroxisome proliferators may depend on pharmacokinetics, relative abundance of PPAR isoforms, nature of PPRE in upstream regions of target genes, the extent of competition or cross-talk among the nuclear transcription factors for the PPAR heterodimerization partner RXR, and the modulating role of coactivators and corepressors on the ligand dependent transcription of PPARs. SRC·I and PBP act as coactivators of PPARs. The binding of SRC-I to PPAR is ligand independent and coexpression of SRC-l with PPAR increases the transcriptional activity of PPAR. PBP also binds to PPAR and this binding is increased in the presence of specific ligands. Since coactivators and corepressors may influence the expression of PPARs, an understanding of the expression patterns of PPARs and other components of transcriptional machinery becomes necessary. Support for a mechanistic relationship between peroxisome proliferation and hepatocarcinogenicity is provided, in part, by a close concordance with the magnitude of hepatic peroxisome proliferation and liver tumor development in rats and mice. Mice deficient in peroxisomal fatty acyl-CoA oxidase, display profound generalized spontaneous peroxisome proliferation and increased mRNA levels of genes that are regulated by PPARa . Hepatic adenomas and carcinomas develop in AOX-I- mice by 15 months of age due to sustained activation of PPARu. These observations implicate acyl-CoA and other putative substrates of AOX as possible ligands for PPARu, thus a normal AOX gene is indispensable for the physiological regulation of PPARu .

84. New Trends in Forensic Toxicology

IS41L 1 I

FORENSIC TOXICOLOGY IN THE RAPIDLY MODERNIZING SOCIETIES OF LATIN AMERICA

A. Wong. Brazil Abstract not received at time of publication.

concentrations and time of onset of drug effects as the intravenous route. In addition, arterial boli drug concentrations reaching the brain are likely to be higher following the smoked route compared to the intravenous route. Overall, these studies demonstrated that the smoked and intranasal routes are highly efficacious for the delivery of illicit drugs and produce a similar profile of drug action to the intravenous route of administration.

IS41L31

RECENT DEVELOPMENTS IN ANALYTICAL TOXICOLOGY, FOR BETTER OF WORSE

Rokus A. de Zeeuw. Dept. ofAnalytical Chemistry and Toxicology, University Centre fo r Pharma cy, NL-9 713 AV Groningen, The Netherlands When considering the state of the art in forensic toxicology from an analytical perspective, the key developments can be subdivided into 3 major areas: (I ) Basic analytical. Here we see the need to be able to deal with more drugs (and metabolites) at ever decreasing levels in a large variety of matrices. As a result, there are constant changes in approaches and instrumentation. (2) Forensic horizon, Gone are the days that forensic toxicology was a rather obscure discipline, mainly limited to digging into dead bodies and affecting only a few people in society. Today. forensic analysis has widened its scope dramatically, in areas such as workplace toxicology, drug abuse testing, drugs and driving, doping, environmental and veterinary toxicology, etc. Hence, it now affects a considerable percentage of our society and focuses much more on living individuals and systems. (3) Validation and interpretation. Because forensic analyses may have severe criminal, civil or punitive consequences, the validation of our methods and approaches is of utmost importance. Also, the interpretation of the analytical results must be done with prudence, taking into account the chemical and biological diversity of our society. The pro' s and con's in these developments will be discussed.

IS4/L41

APPLICATIONS OF ALTERNATIVE MATRICES (HAIR, SALIVA , SWEAT ...) IN FORENSIC TOXICOLOGY

P. Kintz *, B. Lud es. lnstitut de Medecine Legale, Strasbo urg, France

Is4/l2 1RECENT DISCOVERIES IN PHARMACOKINETICS OF DRUGS OF ABUSE

E.J. Cone. Addiction Research Center, NIDA, PO Box 5180, Baltimore, MD, USA Controlled human dosing studies with drugs of abuse have revealed the importance of the chosen route of administration on the delivery of drugs to the bloodstream and to their site of action. Recently, the intranasal and smoked routes have become favored by some populations for the administration of illicit drugs. Research studies with experienced heroin and cocaine users indicated that intranasally administered drug generally provided lower blood concentrations of drug and a slower onset of action compared to the intravenous route; however, intranasal doses are easily manipulated by the user and adequate bioavailability and desired drug effects can be achieved. In addition, the trauma of needle use is avoided and disease exposure is reduced by this route. For marijuana, the smoked route of administration has always been the preferred route. In recent studies with smoked marijuana, it was revealed that single puffs of marijuana smoke produced detectable blood concentrations of tetrahdyrocannabinol, the active ingredient of marijuana. Continued smoking produced rapid increases in blood concentrations with peak concentrations and effects occurring before or near the end of smoking, demonstrating the rapidity and efficacy of the smoking route for marijuan a. The smoked route has also become popular with cocaine and heroin users. This route provided equivalent peak blood

It is generally accepted that chemical testing of biological fluids is the most objective means of diagnosis of drug use. The presence of a drug analyte in a biological specimen can be used to document exposure. The standard in drug testing is the immunoassay screen, followed by the gas chromatographic-mass spectrometric confirmation conducted on a urine sample. In recent years, remarkable advances in sensitive analytical techniques have enabled the analysis of drugs in unconventional biological specimens such as saliva, sweat, meconium or hair. The advantages of these samples over traditional media, like urine and blood, are obvious: collection is almost noninvasive, relatively easy to perform, and in forensic situations it may be achieved under close supervision of law enforcement officiers to prevent adulteration or substitution. Moreover, the window of drug detection is dramatically extended to weeks, months or even years. The aim of this review is to document usefullness of these alternative matrices in forensic situations.