EVALUATION OF PROPINEB EFFECT GENOTOXICITY MONOOXYGENASE ACTIVITIES AND

EVALUATION OF PROPINEB EFFECT: GENOTOXICITY, MONOOXYGENASE ACTIVITIES AND PROPYLENETHIOUREA QUANTIZATION Clara DELLA CROCE, Marco CINI, Leonardo CALTAVUTURO, Giorgio BRONZETTI and Mario GIORGI * IBBA (CNR) – Sezione di Pisa, Via Moruzzi 1 – 56124 Pisa, Italy - * Dipartimento Clinica Veterinaria, Viale delle Piagge 2, 56124 Pisa, Italy INTRODUCTION AIM Pesticides are a large group of substances with different chemical and structural features. Also if these molecules exerted beneficial effects they showed dangerous action on plants, insects and other biological systems. So it is very important to consider that they are biologically active and widespread in the environment. Propineb, a polyvalent fungicide, belonging to dithiocarbamates, is currently used in Italian agricultural practice (fruit, wine and tobacco cultures) and in the industry (wood, paper, plastic, textile manufacture) It belongs to II toxicological class. Oral LD 50 in rats and rabbits are 8500 and 2500 mg /kg respectively. Its chemical structure is similar to maneb and zineb but few data on its effects are available. In vitro studies demonstrated that it was negative in Salmonella typhimurium and in the mouse micronucleus test; a toxicological study in rats showed that propineb caused a high mortality with biochemical, histochemical and ultra-structural changes in liver, brain, thyroid gland, myocardium, spleen and bone marrow. Mutagenesis and genotoxicity of propineb are not yet elucidated and on its main product, propylenethiourea (PLTU) few data are available. EXPERIMENTAL PROCEDURE Genotoxicity of propineb on yeast Saccharomyces cerevisiae. Short term tests on yeast Saccharomyces cerevisiae cells were performed as previously described in literature. Action of propineb on rat and rabbit monooxygenase enzymes in hepatic microsomes after an acute treatment with different doses of pesticide. Cytochrome P-450, aminopyrine-N-demethylase (APD), aniline-N-hydroxylase (An. H), etoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-deethylase (PROD) and western-blot analysis on rat and rabbit hepatic microsomes, treated with different doses of propineb, were determined according to standard protocols. Determination of propylenthiourea (PLTU) in hepatic fractions after a chronic treatment with propineb. Histological modifications of hepatic tissue after acute and chronic treatment. Determination of PLTU concentration, in hepatic fractions treated with propineb, was performed by HPLC Histological modifications of hepatic rat and rabbit tissue, treated with different doses of propineb, after fixation in formalin (10%), inclusion in paraffin (5µm), coloration with hematoxyline ed eosin were evaluated. Statistical analysis was performed by Student’t test for unpaired data (p 0. 05). In vitro and in vivo results are expressed as media of different experiments ±standard deviation. Yeast cytotoxicity 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 B SP % surv. A LP C DMSO 0. 9 1. 8 3. 6 7. 2 14. 4 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 C Determination of Cytochrome P 450 (fig 1), Apd(fig 2), Anh (fig 3) Erod(Fig 4) and Prod (fig 5) in hepatic microsomes of basal rats after a single treatment with a different doses of propineb. 0. 29 Propineb effect on survival on logarithmic (LP) and stationary cells (SP) (panel A) and during the growth ( panel B) in yeast Saccharomyces cerevisiae. RESULTS SHOWED THAT PROPINEB WAS NOT CYTOTOXIC IN SP CELLS AT CONCENTRATIONS RANGING FROM 1 TO 14 mg /m. L, WHILE IN LP CELLS SURVIVAL DECREASED OF ABOUT 65% AT 3 mg /m. L. IN THE SAME EXPERIMENTAL CONDITIONS NO MUTAGENIC ACTION WAS EVIDENCED. WHEN PROPINEB WAS INCUBATED, DURING THE GROWTH, AN IHIBITION OF 100% WAS EVIDENCED AT 0. 29 mg/m. L FIG 2 : IN RATS THE INHIBITORY EFFECT WAS EVIDENT AT ALL DOSES EMPLOYED. IN RABBITS A SIGNIFICANT DECREASE WAS SHOWED ONLY AT 250 mg/kg FIG 1 : IN RATS THE INHIBITORY EFFECT WAS EVIDENT AT 250 AND 500 mg/kg. IN RABBITS A SIGNIFICANT DECREASE WAS SHOWED AT 125 AND 250 mg/kg 0. 029 propineb (mg/ml) DMSO A B C FIG 3: IN RATS THE INHIBITORY EFFECT WAS EVIDENT AT 125, 250 and 500 mg/kg IN RABBITS A SIGNIFICANT DECREASE WAS SHOWED AT ALL DOSES EMPLOYED Observation of Rat (panel A) and Rabbit (Panel B) weight variation during a chronic treatment with propineb RESULTS SHOWED THAT THE DECREASE OF WEIGTH WAS MORE EVIDENT IN RATS WITH RESPECT TO RABBITS PLTU quantization in hepatic homogenate, S 9 - fraction, and microsomes in rats and rabbits treated with propineb (panel C) PLTU WAS DETECTED IN ALL THREE HEPATIC FRACTIONS. IT WAS DIRECTLY PROPORTIONAL TO DOSE EMPLOYED AND DURATION OF TREATMENT FIG 4 : IN RATS THE INHIBITORY EFFECT WAS EVIDENT AT ALL DOSES EMPLOYED. IN RABBITS A SIGNIFICANT DECREASE WAS SHOWED ONLY AT 125 AND 250 mg/kg FIG 5 : IN RATS THE INHIBITORY EFFECT WAS EVIDENT AT ALL DOSES EMPLOYED IN RABBITS A SIGNIFICANT DECREASE WAS SHOWED ONLY AT 250 mg/kg REFERENCES Borgers M et al (1983) Am J Med 74 : 2 -8 Burke MD et al (1985) Biochem Pharmacol 34: 3337 -3345 Hepatic tissue of basal rat (right panel) and after a chronic treatment with propineb, 125 mg/kg for 15 days (left panel) IT WAS EVIDENT THAT PROPINEB CAUSED HISTOLOGICAL DAMAGES Caccialanza G et al (1980) Il Farmaco 35 : 449 -454 Chii-Ling J et Li Gwo-Chen (1980) K’o Hsuch Fa Chan Yueh K’an 8: 551 -559 Del Carratore R et al (1983) Mutat Res 31: 347 -350 Hunter AL and Neal LA (1975)Biochem Pharmacol 24: 2199 -2205 Johnson D J et al (1996) Chem Res Toxicol 9: 910 -916 Ko L et al (1987) Cancer Res 47 : 3101 -31 09 Larsson K S et al (1976) Teratology 14: 171 -134 Lowry 0 et al (1951) J Biol Chem 193: 265 -275 Lubet R A et al (1985) Arch Biochem Biophys 238: 43 -48 Marcireau C et al (1990) Antimicrob Agents Ch 34: 989 -993 Mazel P (1971) In : Fundamental Drug Metabolism and Disposition - La Du BN, Mandel MG. , Way EL (Eds) Williams and Wilkins Company Baltimora 546 -550 Menicagli S et al (1990) Toxicology 64: 141 -153 Morichetti E et al (1992) Toxicol Environ Chem 37: 35 -38 Muccinelli M (1987) In: Prontuario dei fitofarmaci. Ottava edizione. Edagricole, Bologna, 32 Omura T, Sato R (1964) J Biol Chem 239: 2370 -2378 Pohl R J et al (1980) Anal Biochem 107: 150 -155 Rolandi A et al (1984) Mutat Res 135: 193 -197 Sax N (1989) In : Dangerous properties of industrial materials. Van N. Reinhold New York. Seventh Ed 1619 Siebert D et al (1970) Mutat Res 10: 533 -543 Vachkova -Petrova R et al (1991) J Toxicol Clin Exper 11: 407 -416 Zimmermann F K et al (1975) Mutat Res 28: 381 -388 CONCLUSIONS - Propineb was not mutagen in yeast cells. The reduction of cell survival confirmed that cytotoxicity could be due to metabolites formed during the processes of biotran-formation and not to propineb itself. It is indeed well known that LP cells possess high level of cytochrome P 450 and are metabolically active. The inhibition of growth in the presence of propineb could be due to fungicide action, as it was described that antifungal compounds, such as ketoconazole, inhibited ergosterol biosynthesis leading to the arrest of cell proliferation in G 1 phase. - Propineb induced a decrease of cytochrome P 450 content on basal rats and rabbits The effect was confirmed by determination of different monooxygenase activities. The significant decrease in APD for rats and rabbits demonstrated the involvement of 3 A family isoforms during the biotransformation of this compound. Propineb was also able to reduce An. H, EROD and PROD activities that are specific for 2 E 1, 2 A 1 and 2 B 1 isoform respectively. It can be hypothesized that the effect of propineb on monoxygenase enzymes was mainly due to reactive atomic sulfur formed during its biotranformation, that can binds to hepatic macromolecules causing the decrease showed in this work. -Histological modification of hepatic tissue have confirmed literatura data showing that an inactivation or inhibition of cytochrome P 450 could cause cellular morphologial modifications. - HPLC method , modified for our experimental conditions, resulted very effective for the determination of PLTU residues in hepatic tissue.