Streptozocin-induced diabetes in rat: Insulin effects on hyperalgesia and on hepatic metabolism of amitriptyline

90

Poster Session 3P. Toxicokinetic

to BU were in accordance with published data. Additionally, the influences of biochemical parameters, alveolar ventilation and blood: air partition coefficients of BU and BMO on the toxicokinetics of BU and BMO were investigated. Based on the extensive model validation in rodents we assume our model to be suitable for the prediction of disposition and metabolism of BU, BMO, GSH- and haemoglobin-adducts in humans for various scenarios of exposure. By means of the PT model areas under blood concentration-time curves (AUC) resulting from a single 8 hr exposure to 10 ppm BU were calculated for rat, mouse and man: AUC of BU was very similar in rat and mouse; in man it was less than half of the value in rodents. AUC of BMO in mouse was about 1.3 and in man 0.3 times the value obtained for the rat. It was attempted to compare the carcinogenic potential of BU in mice and rats respecting the carcinogenic potentials of both epoxides. However, a satisfactory explanation for the vast differences in the species specific susceptibility could not be obtained. The discrepancy between the estimated and experimentally observed carcinogenic potential might indicate further factors besides BMO and BDI to be involved in the carcinogenicity of BU in rodents.

Keywords: 1,3-butadiene; 1,2-epoxy-3-butene; 1,2:3,4-diepoxybutane; glutathione; haemoglobin adducts; toxicokinetics PERMEATION OF N,N-DIMETHYLFORMAMIDE IP3P-325 f VAPOUR THROUGH SKIN OF RAT AND MAN

Paul E. Kreuzer, Cordula M. Baur i, Winfried Kessler *, Jianhua Shen 2, Johannes G. Filser. GSF-Institutfiir Toxikologie, Neuherberg, Germany; ] lnstitut flir Gerichtsmedizin, LMU Miinchen, Germany; 2 Shanghai Inst. of Entomology, Academia Sinica, Shanghai, China N,N-Dimethylformamide (DMF) is a widely used amphiphilic solvent. The predominant effects upon exposure to DMF are hepatotoxicity in animals and man and embryotoxicity and teratogenicity in animals. From its physicochemical properties a significant dermal uptake of DMF can be expected. The aim of this work was therefore to investigate the kinetics of transdermal absorption of DMF vapour in Sprague-Dawley rats and in humans. Studies were performed in vitro using excised skin of rats and humans and in vivo in rats using a special open system which allowed either dermal or inhalative exposure [1]. In vitro, the amount of DMF permeating through skin samples was determined in an acceptor medium. In vivo, the amount of DMF absorbed dermally was calculated from the DMF vapour concentrations between the inlet and outlet of the exposure chamber. The permeability constant Kp [cm/h], expressed by the ratio of the DMF flux across the skin [/zmol DMF/cm 2 skin/h] and the exposure concentration, was 18 + 8 in 3 dorsal and 2 ventral rat skin samples and 27 -4- 15 in 5 human forearm skin samples obtained during autopsy. Kp in rats in vivo was 51. It can therefore be assumed that Kp for man in vivo will be in the same range. The resulting Kp values of DMF together with in vivo data on the inhalation kinetics of DMF led to the conclusion that the dermal absorption rate of DMF vapour exceeds the rate of inhalative uptake. [1] Kreuzer, PE, GSF-Bericht 19/92, GSF, 1992.

Keywords: dimethylformamide; percutaneous absorption; rat; man

J

COMPARISON OF METABOLIC RATIOS TO ASSESS rP3P.326 r CYPIA2, XANTHINE OXlDASE AND N-ACETYLTRANSFERASE ACTIVITIES USING CAFFEINE AS A METABOLIC PROBE

B. Sinu~s *, A. Ferrdndez, M.A. S~ienz, J. Lanuza, A. Fanlo, G. Laguens I A. Duefias 2. Clinical Pharmacology Service, Universitary Clinic Hospital Zaragoza, Spain; I Emergency Service, Universitary Clinic Hospital Zaragoza, Spain; 2 Clinical Pharmacology and Toxicology Service, "Rio Hortega" Universitary Clinic Hospital, Valladolid, Spain CYPIA2 is a cytochrome P450 involved, as phase I enzyme, in the bioactivation of environmental chemicals to produce active intermediates which may be toxic. Xanthine oxidase (XO) is the best-documented source of oxygen-derived free radicals. Polymorphic N-acetyhransferase (NAT-2) is responsible for the interindividual differences in arylamine-induced bladder cancer. Owing to its ubiquitous use of caffeine have become popular as an in vivo probe for phenotyping CYP1A2, XO and NAT-2 activities. Different metabolic ratios have been proposed to evaluate these activities, based on the involvement of these enzymes in the muhiple caffeine metabolic pathways. In the present work, 148 healthy volunteers, aged 17-57 years, participated in the study (73 smokers and 75 non smokers). 15 metabolites and caffeine were quantified by an HPLC assay. Five metabolic ratios have been compared to assess de inducible CYP1A2 activity. Unimodal distributions were observed with all five metabolic ratios. The ratio: (AFMU + IX + IU)/17U showed to be independent on other enzyme activities. In addition, this ratio resulted in being the best metabolic biomarker of smoking inducibility, which is consistent with the known CYP1A2 transcriptional induction by the binding of PAHs to the AH receptor. Three metabolic ratios have been evaluated to assess NAT-2 activity. The ratio AFMU/(AFMU + 1X + 1U) has been the more accurate to evaluate this polymorphic expression. A close association has been found between lU/(1X + 1U) and lU/1X as indexes for XO activity.

Keywords: CYP1A2; xanthine oxidase; N-acetyltransferase; caffeine test; metabolic ratios

IP3P-3271

STREPTOZOCIN-INDUCED DIABETES IN RAT: INSULIN EFFECTS ON HYPERALGESIA AND ON HEPATIC METABOLISM OF AMITRIPTYLINE

Francois Coudor6 *, Christine Courteix, Roy Massingham t, Joseph Fialip. Laboratoire de Pharmacologie, Facultd de Pharmacie, 63001 Clermont-Ferrand cedexl, France; i Riom Laboratoires-CERM 63203 Riom, France Streptozocin (STZ), a toxic drug for pancreatic Langerhans islets, is used to induce experimental insulin-dependent diabetes in rats, which show altered pain sensitivity, as hyperalgesia and allodynia [1]. These symptoms are relatively resistant to antidepressants such as amitriptyline (AMI), which are effective to relieve painful neuropathies in diabetic patients [2]. The purpose of this study was to estimate the effect of insulin on (i) diabetes-induced hyperalgesia in STZ-diabetic rats and (ii) AMI metabolism in hepatocytes from STZ-diabetic rats. Animals with a glycaemia >_ 2.5 g/l (14 mM) were considered to be diabetic. Each day, mechanical hyperalgesia was in vivo assessed in STZ rats by using the paw-pressure test (threshold: pawwithdrawal). Insulin (Insuline Lente MC, 40 UI/ml, Novo Nordisk) was daily subcutaneously (93 to 153 nmol/kg) injected until normalization of glycaemia. In vitro systems were prepared according to the method of Moldeus. Analysis of AMI and both demethylated and hydroxylated metabolites was carried out by an HPLC method [3] at various times of incubation of STZ-diabetic hepatocytes with a non-cytotoxic AMI level (10/zg/ml) and 0.075% insulin.

Poster Session 3P Toxicokinetic Results show: i) daily injections of insulin progressively reverse hyperalgesia parallel to the normalization of glycaemia, ii) deviations of AMI metabolism in diabetic hepatocytes, i.e. increase of demethylation reactions and decrease of hydroxylation ones, not corrected by insulin addition. These data suggest that hyperglycaemia induces (i) peripheral and central nervous system alterations which are reversible by insulin treatment and (ii) modifications of hepatic metabolism resistant to insulin. This may result from damage induced by STZ on some CYT P450 enzymes, and suggest that STZ per se may not have any direct toxic effect on peripheral and central system. [1] Courteix C et al., Pain, 53: 81-8, 1993, [2] Courteix C et al., Pain, 57: 153-60, 1994. [3] Coudor6 F et al., J. Chromatogr., 58: 249-55, 1992.

Keywords: streptozocin; toxicity; diabetic rats; insulin; pain; in vitro

91

metabolite, norcocaine. To find out which CYP enzymes are candidates to metabolize cocaine, human testosterone 6fl-hydroxylase and coumarin 7-hydroxylase were incubated with both cocaine and norcocaine. Norcocaine inhibited more potently testosterone 6~hydroxylase (IC50 0.8 mM) than cocaine (IC50 > 1 mM) and at 10 mM concentration both inhibited coumarin 7-hydroxylase only by about 30%. Norcocaine induced type I substrate binding spectra with solubilized microsomes in t h e / z M concentration range but type II spectra when the concentration was increased to the 100/zM range. Incubation with NADPH led to the formation of two still unidentified HPLC-peaks from norcocaine. We conclude that norcocaine is bound to CYP enzymes and further metabolized by the same enzymes.

Keywords: monooxygenase; cocaine; norcocaine; inhibition

P3P-330 I[ INDUCTION OF HEPATIC ENZYME ACTIVITY IN RAT )

BY POLYCYCLIC AROMATIC HYOROCARBONS

P3P-3281

SEX-DEPENDENT RESPONSES OF MOUSE HEPATIC CYP ENZYMES TO NORCOCAINE

Anu Tervo * 1, Risto Juvonen m,Pertti Pellinen i Frej Stenback 2, Markku Pasanen 3,4.1 Department of Pharmacology & Toxicology,

University of Kuopio, Finland; 2Department of Pathology, University of Oulu, Finland; 3Department of Pharmacology & Toxicology, Universityof Oulu, Finland; 4 Central Laboratory of University Hospital of Oulu, Finland Earlier studies have shown that CYP3A enzymes in mouse and human liver microsomes catalyze the conversion of cocaine to norcocaine (NOR), and that male mouse is more susceptible to cocaine-induced hepatotoxicity than female. In this study we examined the effects of a single dose of NOR on hepatic CYP enzymes in male and female DBA/2 mice. Significant sex-dependent differences were observed: In females NOR (< 60 mg/kg) did not cause hepatotoxicity as detected by histopathology or serum alanine aminotransferase (S-ALAT) determinations. In males with NOR dose 40-60 mg/kg a dramatic increase in S-ALAT was observed; up to 2000 U/I of S-ALAT activity correlated with microsomal coumarin 7-hydroxylase activity (COH) while at higher S-ALAT activities, COH became decreased. In females COH was slightly enhanced with increased NOR doses but no increase in ALAT was observed. In a time course study, a single dose of 60 mg/kg NOR increased COH marginally in females for three days. Moreover, in females testosterone 6/~-hydroxylase activity was increased by 2-3 fold with doses of 30 mg/kg but in males by 1.5 fold. Testosterone 15ot-hydroxylase was enhanced in females until the fifth posttreatment day while in males a permanent decrease was observed already 12 h after treatment. These results demonstrate that sex-dependent metabolic interactions of cocaine with CYP enzymes are not only restricted to the parent compound but may also be detected - even more profoundly - with its metabolite norcocaine.

Keywords: hepatotoxicity; cocaine; norcocaine; monooxygenase

IP3P-3291

INTERACTIONS OF COCAINE AND NORCOCAINE WITH HUMAN LIVER MICROSOMAL MONOOXYGENASE ENZYMES IN VITRO

Liisa Kulmala * i, Markku Pasanen 2, Pertti Pellinen 1, Esko Alhava 3, Risto Juvonen i. i Department of Pharmacology and

Toxicology, Universityof Kuopio, POB 1627, 70211 Kuopio, Finland; 2Department of Pharmacology and Toxicology, University of Oulu, Central Laboratory, University Hospital of Oulu, Finland; 3Department of Surgery, University ofKuopio, POB 1627, 70211 Kuopio, Finland Our previous studies have shown that human CYP3A enzymes catalyze the oxidative metabolism of cocaine producing a toxic

Malgorzata Gawlik. Jagiellonian University, Collegium Medicum,

Department of Toxicology, Cracow,Poland The hepatic cytochrome P450s are mixed-function oxidases which metabolize a wide variety of xenobiotics, and also bioactivate carcinogens such as polycyclic aromatic hydrocarbons to reactive metabolites. To investigate possible relationship between cytochrome P450 induction and clearance of pyrene male Wistar rats were p.o. treated with benzo/ghi/perylene (20 mg/kg) in corn oil, once daily for 3 days. Controls received oil only. Pyrene (20 mg/kg) suspended in Tween 80: saline (1 + 9) was given i.v. and p.o. on 4 consecutive days. Blood samples from carotid artery were taken from 0.25 to 6 hours and concentration of unchanged pyrene was determined by gas chromatography method. Toxicokinetic parameters such as the biological half-life, volume of distribution, clearance, area under the concentration-time curve were calculated [1]. To enzyme analysis, individual livers were homogenized and the activity of 7ethoxycoumarin O-deethylase was measured in microsomal samples [2]. This study shows that three daily treatment of benzo(ghi)perylene do not change significant toxicokinetic parameters of pyrene. 7hydroxycoumarin O-deethylase activity in liver microsomes was determined as a function of protein concentration. Enzyme activities in liver microsomes of benzo(ghi)perylene-treated rats were significant higher compared to controls. Observation of the inductive activity of benzo(ghi)perylene indicate that various isozymes play role in biotransformation of pyrene and benzo(ghi)perylene. [1] Lipniak M., Polycyclic Aromatic Compounds, 3, 111-119, 1993 [2] Rosenberg D.W., Anal. Biochem., 191,354-358, 1990

Keywords: polycyclic aromatic hydrocarbons; toxicokinetic; 7ethoxycoumarin O-deethylase activity; rat

[ P3P-331 I ONTOGENESISOF HEPATIC BIOTRANSFORMATION PROCESSES IN RAT MALES

Andrzej Plewka *, Marcin Kamirlski, Danuta Plewka. Silesian

School of Medicine, Departmentof Histology & Embryology, Katowice, Poland The cytochrome P-450-dependent monooxygenase system is responsible for liver biotransformation of endo- and endogenous substances. The activity of monouxygenases is regulated and modulated by many factors, including age. In this study we decided to determine relationships between cellular metabolism of the hepatocyte and age via evaluating hydroxylation and N-demethylation of xenobiotics. The study was performed on several age groups of male Wistar rats, namely very young, sexually mature, ageing (20 months), and very old rats (28 months). Using standard methodology, we evaluated