Effect of Ivermectin on Induction of ytochromes P450 in Mle Rts L. SKÁLOVÁ, B. SZOTÁKOVÁ*, M. MAHALA, J. NEA, b J. LAMKA, b L. DUHÁEK, nd E. KVASNIKOVÁ Deprtment of Biochemicl Sciences, Fculty of Phrmcy, hrles University, Z-500 05 Hrdec Králové b Deprtment of Phrmcology nd Toxicology, Fculty of Phrmcy, hrles University, Z-500 05 Hrdec Králové c Veterinry Reserch Institute, Z-621 32 Brno Received 18 October 1999 Effects of the widely used ntiprsitic drug ivermectin on cytochromes P450 were tested in dult mle rts. Liver microsomes were prepred from nimls fter single or repeted tretments with vrious doses of ivermectin. The heptic microsoml protein content nd ctivities of cytochromes P450 towrds lkoxyresorufins nd testosterone s the specific substrte probes were mesured nd the results were compred with the dt obtined in control nimls. The ^егаред^с dose cused only nonsignificnt increse of the content of microsoml proteins nd the ctivities under study, while the tretment with the higher doses led to significnt increses of the concentrtions of microsoml proteins nd ctivities of 7-methoxyresorufin O-demethylse nd 7-ethoxyresorufin 0deethylse. The results indicte the potentil of ivermectin to induce the expression of cytochrome P4501A isoenzymes, which re known to be the biotrnsformtion enzymes responsible for metbolic ctivtion of promutgenes. The generic nme ivermectin hs been ssigned to the mss mixture of the 22,23-dihydrovermectines rbbi nd H-2Bib (wt = 80 : 20) used widely s n ntiprsitic drug with brod spectrum of ctiv ity ginst severl species of nemtodes, rchnids, nd insects prsitizing in frm nimls, cloven-hoofed gme, nd severl other species of nimls [1]. The ivermectin is dministered in nimls prenterlly (in jections, pour-on solutions) or orlly (grnulte, pste, tblet). Bsed on the type of dosge form single or re peted doses re used. Its world-wide cceptnce in helth cre of food nd compnion nimls hs mde it mjor commercil success. The ivermectin hs lso been found to be n effective microfilricidl gent in humn medicine [2]. The presence of drug residues in food products of niml origin depends on phrmcologicl properties of the drugs s well s on the physiologicl specificities of the species nd breed of food nimls [3]. Our pre vious mesuring of residul concentrtions in clovenhoofed gme treted orlly with ivermectin yielded rther unexpected result in tht the withdrwl period fter repeted dministrtion of therpeutic doses (6 dys) ws shorter in comprison with single dmin istrtion [4]. To elucidte these findings, our investi gtions were focused on possible effects of ivermectin on ctivities of drug-metbolizing enzymes, since the length of the withdrwl period is determined minly by the drug biotrnsformtion pthwys. The ctiv ities of the biotrnsformtion enzymes cn be influ enced by mny fctors, the most importnt of which is enzyme induction, i.e. n increse in the expres sion of the gene encoding the enzyme s response to the presence of xenobiotic in the orgnism [5 7]. Ivermectin is highly lipophilic substnce with long hlf-time period. This property indictes the possibil ity tht ivermectin cts s n inducer of biotrnsfor mtion enzymes. The induction or suppression of drug-metbolizing enzymes is one of the importnt consequences of d ministrtion of phrmceuticls. Possible induction should be tested crefully in ll drugs prior to their relese for clinicl testing. hnges in ptterns of drug biotrnsformtion isozymes my essentilly lter physiologicl processes nd phrmcologicl or toxicologicl impcts of mediction or exposure to envi ronmentl contminnts [7, 8]. ytochromes P450 ply the key role in the bio trnsformtion of ivermectin [9]. In this study, 7lkoxyresorufins nd testosterone were used s the *The uthor to whom the correspondence should be ddressed. hem. Ppers 54 (4)249 253 (2000) 249
L. SKÁLOVÁ, B. SZOTAKOVA, M. MAHALA. J. NEČA, J. LAMKA. L. DUHAEK, E. KVASNIKOVÁ o 'o. H 3 OH rboi НгВ1ь R H2H3 R = H3 specific substrte probes to ssess the possible influence of ivermectin dministered in therpeutic or excessive doses on the ctivities of cytochrome P450 isozymes in rts. EXPERIMENTAL Ivermectin (IVOME inj., 50 cm 3, H 30490) ws purchsed from MSD Agvet, Prgue, zech Republic. 7-Methoxy-, 7-ethoxy-, 7-pentoxy-, nd 7- benzyloxyresorufins were obtined from Moleculr Probes (Eugene, MI, USA), nd the other specific substrtes nd products were supplied by Sigm. All the other chemicls were of the highest purity vilble commercilly. Animls nd Preprtion of Subcellulr Frctions Mle dult rts (Rttus norvegicus vr. lb, mss pproximtely 275 g) were obtined from the Reserch Institute for Phrmcy nd Biochemistry (Prgue, zech Republic). The 16 nimls were divided into four groups. The control group received six doses of wter, group A ws treted with single dose of 35 mg of ivermectin in wter suspension per 1 kg of rt body mss (35 mg kg -1 ), group В with six therpeutic doses of 0.3 mg of ivermectin in wter suspension per 1 kg of rt body mss (0.3 mg kg -1 ), nd group С with six successive doses of 10 mg of ivermectin in wter suspension per 1 kg of rt body mss (10 mg kg -1 ). All the nimls were treted orlly, fed on stndrd diet with free ccess to drinking devices, fsted 12 h before the end of the experiment, nd scrificed by decpittion under ether nesthesi. Livers were collected nd homogenized t p = 0.3 g cm -3 (1 g of liver tissue per 3 cm 3 of buffer) in 0.1 M sodium phosphte buffer, ph 7.4, using the Potter Elvehjem homogenizer. The microsoml frction ws seprted by ultrcentrifugtion of the liver homogente s described [10, 11]. Enzyme Assys The 7-ethoxyresorufin (EROD), pentoxyresorufin (PROD), methoxyresorufin (MROD), nd benzyloxyresorufin (BROD) O-delkylse ctivities were determined using fluorimetric determintion of resorufin [12] t 30 with the finl concentrtions of the substrtes 2 /xmol dm -3. The ssys were conducted using the Perkin Elmer luminescence spectrophotometer LS 50B with the excittion nd emission wvelengths of 530 nm nd 585 nm, respectively. The EROD, PROD, MROD, nd BROD ctivities were clculted using the stndrd mount-ddition technique. The heptic microsoml testosterone hydroxylse (ТОН) ctivities were ssyed using essentilly the method ccording to [13]. The rection mixture (totl volume 1 cm 3 ) contined 0.1 cm 3 of microsomes, 10 mm 3 of solution of testosterone in methnol (c = 25 mmol dm -3 ), 10 mm 3 of NADPH (103 mg cm -3 wter) in 0.01 M phosphte buffer, ph 7.5. After 15 min of incubtion t 37, the rection ws stopped by the ddition of 6 cm 3 of dichloromethne. After shking 250 hem. Ppers 54 (4) 249 253 (2000)
EFFET OF IVERMETIN IN RATS 16 MROD EROD PROD BROD Fig. 1. Specific YP ctivities \ towrds 7-lkoxyresorufins in heptic microsomes of control or ivermectin-treted rts (A, single dose 35 mg kg -1 ; B, six successive doses of 0.3 mg kg -1 ;, six successive doses of 10 mg kg -1 ). Activities expressed s \ = (n(resorufin)/(m(protein) t))/(pmo\ mg -1 min -1 ), n = 4, *p < 0.05, **p < 0.01. nd centrifugtion, the orgnic phse ws seprted nd dried. The smples were dissolved in methnol wter solution (ip r = 1:1) nd the products were nlyzed using Wters HPL system (NovPk 18 column, 4 /xm). The mobile phse consisted of 20 % methnol in 0.04 % cetic cid (A) nd methnol with 0.04 % cetic cid (B); the grdient of the mobile phses ws 15 40 % В in 33 min. Protein concentrtions were ssyed using the bicinchoninic cid method [14]. RESULTS AND DISUSSION Long-term or repeted interctions of drugs or other xenobiotics with live orgnisms cn result in the induction of enzymes, prticulrly those prticipting in the metbolism of the inducing gent. Investigtions of the metbolism of ivermectin in microsomes of rts nd steers [15] nd swine [9] hve identified 24- hydroxymethyl nd de-3-0-methyl derivtives s the mjor metbolites. The structure of ivermectin nd its mjor metbolites let us ssume tht cytochromes P450 (YP) ply the key role in its metbolism. ytochrome P4503A4 (YP3A4) hs been identified s the predominnt enzyme responsible for the metbolism of ivermectin in humn liver microsomes lie]. The induction of the YP isoenzymes cn be ssessed by mesuring the enzyme ctivities towrds specific substrtes in vitro. In this study the effects Tble 1. The Proteins ontent in Rt Microsomes from ontrol Animls or Ivermectin-Treted Animls Rt group p*(microsoml proteins) ± SD/(mg cm -3 ) ontrol A В С 8.98 ± 0.85 9.93 ± 0.97 7.88 ± 0.60 11.20 ± 0.84 * Averge vlue of four experiments. A = single dose 35 mg kg -1, В = repeted doses б x 0.3 mg kg -1, С = repeted doses 6 x 10 mg kg -1. of orl dministrtion of ivermectin on heptic microsoml YP ctivities were investigted in control rts nd in rts treted with single dose (group A), repeted therpeutic doses (group B), nd repeted 30-fold higher doses (group ). Microsoml protein concentrtions were determined s rough prescreening mrker of induction. A significnt increse of the protein content ws found in microsomes of nimls of group С treted repetedly with 10 mg of ivermectin per 1 kg of rt body mss (Tble 1). The ctivities towrds substrtes reltively specific for YP isoenzymes were studied to evlute possible effects of ivermectin on cytochromes P450. The following specificity ptterns hve been found for 7-lkoxyresorufins used s substrte probes for the determintion of ctivities of individul YP isoen- hem. Ppers 54 (4) 249 253 (2000) 251
L. SKÁLOVÁ, B. SZOTÁKOVÁ, M. MAHALA. J. NEČA, J. LAMKA, L. DUHÁČEK, E. KVASNIČKOVÁ 1200 6b-TOH 16-TOH Fig. 2. Heptic microsoml testosterone hydroxylse ctivities ß\ in control nd ivermectin-treted rts. 6b-TOH, testosterone б/3-hydroxylse ctivity; 16-TOH, testosterone 16-hydroxylse ctivity; for tretment nd other bbrevitions see Fig. 1. Activities expressed s ß\ = (n(product)/(7n(protein) č))/(pmol rng" 1 m i n - 1 ), n = 4. zymes [17, 18]: E R O D is specific for Y P 1 A 1 nd less for YP1A2; M R O D is specific for YP1A2 nd less for Y P 1 A 1 ; P R O D is specific for Y P 2 B isoen zymes; B R O D is specific for Y P 3 A nd prtly for Y P 1 A 1 nd YP1A2. T h e r p e u t i c doses of iver mectin in r t s induced only nonsignificnt increse of E R O D nd prtly lso t h e B R O D ctivities, but the dministrtion of the higher doses in groups A nd С resulted in significnt induction of the M R O D nd E R O D ctivities. T h e induction p t t e r n of 0delkylse ctivities is presented in Fig. 1. Heptic microsoml testosterone hydroxyltions were not significntly influenced by ivermectin (Fig. 2). Testosterone б/3-hydroxylse nd lg-hydroxylse c tivities hve been reported to be minly ssocited with the YP3A nd Y P 2 enzymes, respectively [19]. Our results indicte t h e bility of high doses of ivermectin t o stimulte t h e expression of the YP1A, but not of t h e Y P 2 B or YP3A forms in rts. y tochromes P4501A re the mjor isozymes involved in the biologicl ctivtion of vrious environmentl pol lutnts including procrcinogens, such s poly cyclic romtic hydrocrbons nd rylmines [7]. T h e in duction of Y P 1 A is obviously ssocited lso with complex of other toxic effects, such s ctivtion of protein kinse с cscde nd incresed mitosis nd cell prolifertion [7, 20]. Therefore, exposure to high doses of ivermectin my increse the risk of mutgenesis nd cocrcinogenesis. However, ny extrpoltion of the d t obtined in r t s to cttle nd deer must be r t h e r reserved tking into considertion species-specific dif ferences in the quntity, ctivity, substrte specificity, 252 nd inducibility of biotrnsformtion enzymes [8, 21]. Moreover, very little is known currently of the chr cteristics of biotrnsformtion enzymes nd their inducibilities in r u m i n n t s [3]. Anyhow, our results cn be regrded s wrning t h t ivermectin cn induce Y P ctivities, nd underline the utility of continuing nlogous studies in trget niml species. Acknowledgements. This work ws supported by the Grnt Agency of the hrles University, Grnt No. 196/97 nd by the Ministry of Agriculture (VRI 306-09). REFERENES 1. hiu, S. L. nd Lu, A. Y. H., Ivermectin nd Abmectin. (mpbell, W., Editor.) Pp. 131 144. Springer-Verlg, New York, 1989. 2. Ette, E. I., Thoms, W. O., nd Achumb, J. I., DIP 24, 426 (1990). 3. Fink-Gremmels, J. nd Vn Miert, A. S. J. P. A. M., Anlyst 119, 2521 (1994). 4. Lmk, J., Her, A., Pešk, R., nd Frglová, К., Sborník referátu, 179 (1994). 5. Jkoby, W. B. (Editor), Enzymtic Bsis of Detoxiction. Acdemie Press, London, 1980. G. Test, В. nd ldwell, J. (Editors), The Metbolism of Drugs nd Other Xenobiotics. Biochemistry of Redox Rections. Acdemic Press, London, 1995. 7. Prke, D. V., Ionnides,, nd Lewis, D. F. V., Toxi col in Vitro 4, 680 (1990). 8. Boobis, A. R., Sesrdic, D., Murry, B. P., Edwrds, R. J., Singleton, A. M., Rich, K. J., Murry, S., De L Torre, R., Segur, J., Pelkonen, O., Psnen, M., Kobyshi, S., Zhi-Gung, Т., nd Dvies, D. S., Xenobiotic 20, 1139 (1990). hem. Ppers 54(4)249 253 (2000)
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