Pathologic Changes in the Liver During Acute Exposure to Clozapine and Its Combination with Ethanol (Experimental Study)

Purpose: identification of pathological changes in the liver at early stages of the effect of clozapine and clozapine combined with ethanol to improve the diagnosis of this acute exposure.

Materials and methods. The experiments were carried out on outbred male rats (n=15). The animals were split into 3 groups, 5 animals in each: 1) control (intact rats); 2) clozapine, 3) clozapine and ethanol. Clozapine at a dose of 150 mg/kg of the animal’s body weight was administered orally under anesthesia (chloralose), clozapine (150 mg/kg) with ethanol at a dose of 5 ml/kg — orally. Rats were removed from the experiment after 3 hours by decapitation. Samples of liver tissue were studied using light microscopy. During histological examination, the following morphological signs were evaluated: blood circulation disorder (presence of venous, capillary, and arterial congestion, appearance of venous hemorrhage), absence of nuclei staining, cytoplasm lumpiness, cytoplasm vacuolation, presence of necrosis foci, appearance of cellular response (a large quantity of leukocytes per power field). The assessment was done using the F-test. Forensic chemical analysis was carried out using a high-performance liquid chromatograph equipped with a mass detector. The content of clozapine, norclozapine, and clozapine-N-oxide in blood serum and liver homogenate of rats was evaluated.

Results. In the control group, no pathological changes in liver tissue were observed. 3 hours after clozapine administration, venous congestion was detected. 3 hours after administration of clozapine and ethanol combination, venous congestion and absence hepatocyte nuclei staining were observed. It was found that after combined administration of clozapine and ethanol, the blood serum clozapine was 1.94-fold and clozapine-N-oxide was 2.3-fold lower than when clozapine was administered as a single drug while the content of norclozapine showed no difference.

Conclusion. Changes detected in the liver, together with changes in other organs and findings of the forensic chemical analysis, allow establishing the fact of clozapine poisoning. Understanding of the processes going on in the body during such intoxications helps rendering medical assistance to victims more effectively.

1. Livanov G.A., Batotsyrenov B.V., Vasilyev S.A., Andrianov A.Yu., Baranov D.V., Nezhentseva I.V. Oxidative distress and its correction with reamberin in patients with acute poisoning by a mixture of psychotropic substances. Obshchaya Reanimatologiya = General Reanimatology. 2013; 9 (5): 18–23. DOI: 10.15360/1813-9779-2013-5-18. [In Russ., In Engl.]

2. VIDAL directory. Medicines in Russia. 21-st ed. Moscow: AstraFarmServis; 2014: 8. ISBN: 978-5-89892-165-1. [In Russ.]

3. Mashkovsky M.D. Medicinal products. 16-th ed. Moscow: Novaya Volna; 2014: 73–74. ISВN 978-5-7864-0218-7. [In Russ.]

4. Burns M.J. The pharmacology and toxicology of atypical antipsychotic agents. J. Toxicol. Clin. Toxicol. 2001; 39 (1): 1–14. DOI: 10.1081/CLT100102873. PMID: 11327216

5. Essali A., Al-Haj Haasan N., Li C., Rathbone J. Clozapine versus typical neuroleptic medication for schizophrenia. Cochrane Database Syst. Rev. 2009; 1:CD000059. DOI: 10.1002/14651858.CD000059.pub2. PMID: 19160174

6. Asenjo Lobos C., Komossa K., Rummel-Kluge C., Hunger H., Schmid F., Schwarz S., Leucht S. Clozapine versus other atypical antipsychotics for schizophrenia. Cochrane Database Syst. Rev. 2010; 11: CD006633. DOI: 10.1002/14651858.CD006633.pub2. PMID: 21069690

7. Stroup T.S., Gerhard T., Crystal S., Huang C., Olfson M. Comparative effectiveness of clozapine and standard antipsychotic treatment in adults with schizophrenia. Am. J. Psychiatry. 2016; 173 (2): 166–173. DOI: 10.1176/appi.ajp.2015.15030332. PMID: 26541815

8. Barber S., Olotu U., Corsi M., Cipriani A. Clozapine combined with different antipsychotic drugs for treatment-resistant schizophrenia. Cochrane Database Syst. Rev. 2017; 3: CD006324. DOI: 10.1002/14651858.CD006324.pub3. PMID: 28333365

9. Dain J.G., Nicoletti J., Ballard F. Biotransformation of clozapine in humans. Drug Metab. Dispos. 1997; 25 (5): 603–609. PMID: 9152600

10. Kaletina N.I. (ed.). Toxicological chemistry. Metabolism and toxicant analysis. Moscow: GEOTAR-Media; 2008: 1016. ISBN 978-5-97040613-7. [In Russ.]

11. Zhou S.F., Zhou Z.W., Yang L.P., Cai J.P. Substrates, inducers, inhibitors and structure-activity relationships of human Cytochrome P450 2C9 and implications in drug development. Curr. Med. Chem. 2009; 16 (27): 3480–3675. DOI: 10.2174/092986709789057635. PMID: 19515014

12. Fang J. Metabolism of clozapine by rat brain: the role of flavin-containing monooxygenase (FMO) and cytochrome P450 enzymes. Eur. J. Drug Metab. Pharmacokinet. 2000; 25 (2): 109–114. DOI: 10.1007/ BF03190076. PMID: 11112091

13. Hsuanya Y., Dunford H.B. Oxidation of clozapine and ascorbate by myeloperoxidation. Arch. Biochem. Biophys. 1999; 368 (2): 413–420. DOI: 10.1006/abbi.1999.1328. PMID: 10441395

14. Slyundin D.G., Livanov A.S., Anuchin V.V., Bobrinskaya I.G., Gutova E.V. Criminal clozapine intoxications. Anesteziologiya i Reanimatologiya. 2007; 4: 61–64. PMID: 17929492. [In Russ.]

15. Ilyashenko K.K., Luzhnikov E.A., Belova M.V., Ermokhina G.V., Lisovik Zh.A., Kareva M.V., Elkov A.N., Zimina L.N., Barinova M.V. Features of acute poisonings by Klozapin. Toksikologichesky Vestnik. 2009; 2: 2–5. [In Russ.]

16. Bogomolova I.N., Bogomolov D.V. Thanatogenesis in poisoning with psychic drugs. Sudebno-Meditsinskaya Ekspertiza. 2005; 48 (2): 19–22. PMID: 15881137. [In Russ.]

17. Coodin S., Ballegeer T. Clozapine therapy and pulmonary embolism. Can. J. Psychiatry. 2000; 45 (4): 395. PMID: 10813078

18. Hägg S., Spigset O., Söderström T.G. Association of venous thromboembolism and clozapine. Lancet. 2000; 355 (9210): 1155–1156. DOI: 10.1016/S0140-6736(00)02066-3. PMID: 10791380

19. Barsegyan S.S., Nikolaeva N.O., Onishchenko M.M., Salomatin E.M., Salnikova E.A. The determination of clozapine for the forensic chemical study of cadaveric blood, urine, and liver with the use of high performance liquid chromatography. Sudebno-Meditsinskaya Ekspertiza. 2012; 55 (4): 43–47. PMID: 23008960. [In Russ.]

20. Sokolova O.I., Malkova T.L., Nevolin N.I. Detection of azaleptine in forensic chemical examination of cadaveric material. Sudebno-Meditsinskaya Ekspertiza. 2007; 50 (2): 35–38. PMID: 17520906. [In Russ.]

21. Baldessarini R.J., Centorrino F., Flood J.G., Volpicelli S.A., Huston-Lyons D., Cohen B.M. Tissue concentration of clozapine and its metabolites in the rat. Neuropsychopharmacology. 1993; 9 (2): 117–124. DOI: 10.1038/ npp.1993.50. PMID: 8216694

22. Subramanian S., Völlm B.A., Huband N. Clozapine dose for schizophrenia. Cochrane Database Syst. Rev. 2017; 6: CD009555. DOI: 10.1002/ 14651858.CD009555.pub2. PMID: 28613395

23. Repetto M.R., Repetto M. Habitual, toxic, and lethal concentrations of 103 drugs of abuse in humans. J. Toxicol. Clin. Toxicol. 1997; 35 (1): 1–9. DOI: 10.3109/15563659709001158. PMID: 9022645

24. Zimina L.N., Mikhailova G.V., Barinova M.V., Pavlenko E.Yu., Polozov M.A., Popov S.V., Rozumnyi P.A., Ilyashenko K.K., Ermokhina T.V. Morphological aspects of acute intoxication with Azaleptin. Sudebno-Meditsinskaya Ekspertiza. 2008; 51 (3): 8–10. PMID: 18589664. [In Russ.]

25. Brown C.A., Telio S., Warnock C.A., Wong A.H. Clozapine toxicity and hepatitis. J. Clin. Psychopharmacol. 2013; 33 (4): 570–571. DOI: 10.1097/JCP.0b013e3182946586. PMID: 23764687

26. Douros A., Bronder E., Andersohn F., Klimpel A., Thomae M., Sarganas G., Kreutz R., Garbe E. Drug-induced liver injury: results from the hospitalbased Berlin Case-Control Surveillance Study. Br. J. Clin. Pharmacol. 2015; 79 (6): 988–999. DOI: 10.1111/bcp.12565. PMID: 25444550

27. 27. Zlatkovic´ J, Todorovic´ N., Tomanovic´ N., Boškovic´ M., Djordjevic´ S., Lazarevic´-Pašti T., Bernardi R.E., Djurdjevic´ A., Filipovic´ D. Chronic administration of fluoxetine or clozapine induces oxidative stress in ratliver: a histopathological study. Eur. J. Pharm. Sci. 2014; 59: 20–30. DOI: 10.1016/j.ejps.2014.04.010. PMID: 24768740

28. 28. Tucker P. Liver toxicity with clozapine. Aust. N.Z. J. Psychiatry. 2013; 47 (10): 975–976. DOI: 10.1177/0004867413488224. PMID: 23636912

29. Wu Chou A.I., Lu M.L., Shen W.W. Hepatotoxicity induced by clozapine: a case report and review of literature. Neuropsychiatr. Dis. Treat. 2014; 10: 1585–1587. DOI: 10.2147/NDT.S67654. PMID: 25210451

30. Romanova O.L., Stepanova E.S., Barsegyan S.S., Sundukov D.V., Chistyakov V.V. Simultaneous determination of clozapine, norclozapine, clozapine-noxide in serum and organs. Farmatsiya. 2016; 65 (5): 27–29. [In Russ.]

31. Elmorsy E., Elzalabany L.M., Elsheikha H.M., Smith P.A. Adverse effects of antipsychotics on micro-vascular endothelial cells of the human bloodbrain barrier. Brain Res. 2014; 1583: 255–268. DOI: 10.1016/j.brainres.2014.08.011. PMID: 25139421

32. Shulpekova Yu.O. Liver drug injury. Consilium medicum. 2006; 8 (7): 5–7 [In Russ.]

33. Orhan H. Extrahepatic targets and cellular reactivity of drug metabolites. Curr. Med. Chem. 2015; 22 (4): 408–437. DOI: 10.2174/0929867321666140826113716. PMID: 25174932

34. Glushkov S.I., Kutsenko S.A., Karpishchenko A.I., Novikova T.M. Status of glutathione system and lipide peroxidation processes in erythrocytes with patients in the clinic of acute poisonings by substances of sedative and hypnotic action. Toksikologichesky Vestnik. 2003; 5: 6–12. [In Russ.]