Zidovudine-induced mitochondrial disorder with massive liver steatosis, myopathy, lactic acidosis, and mitochondrial DNA depletion

Copyright © European Association .for the Stu@ of the Liver 1999

Journal of Hepatology 1999; 30:156-160 Printed in Denmark . All rights reserved Munksgaard" Copenhagen

Journal of Hepatology ISSN 0168-8278

Case R e p o r t

Zidovudine-induced mitochondrial disorder with massive liver steatosis, myopath3 lactic acidosis, and mitochondrial DNA depletion Patrick Chariot 1'2'4, Ir6ne Drogou 4, Isabelle de Lacroix-Szmania 3, Marie-Christine Eliezer-Vanerot 4, B6n6dicte Chazaud 4, Anne Lomb6s 5, Annette Schaeffer 3 and Elie Serge Zafrani 1 Departments of IPathology, 2Toxicology. and 3Internal Medicine, Hdpital Henri Mondor, Crdteil; the 4Groupe d'Etudes et de Recherches sur le Muscle et le Nerf (EA 2347, UniversitO Paris XII), FacultO de Mddecine, CrOteil: and 5INSERM U 153. Paris, France

Zidovudine is known to be responsible for a mitochondrial myopathy with ragged-red fibres and mitochondrial D N A depletion in muscle. Lactic acidosis alone or associated with hepatic abnormalities has also been reported. A single report mentioned the concomitant occurrence of muscular and hepatic disturbances and lactic acidosis in a patient receiving zidovudine, but muscle and liver tissues were not studied. A 57-yearold man with AIDS, who had been treated with zidovudine for 3 years, developed fatigue and weight loss. Serum creatine kinase and hepatic enzyme levels were high. Lactic acidosis was present. Liver biopsy showed diffuse macrovacuolar and microvacuolar steatosis. After withdrawal of zidovudine, creatine kinase, aspartate aminotransferase, and alanine aminotransferase levels normalised within 5 days, and lactacidae-

mia decreased. Acidosis persisted. The patient became confused and febrile and died 8 days after detection of high blood lactic acid. A muscle sample obtained at autopsy showed mitochondrial abnormalities with ragged-red fibres and lipid droplet accumulation. Southern blot analysis showed depletion of mitochondrial DNA, affecting skeletal muscle and liver tissue. No depletion was found in myocardium and kidney. This case emphasises that zidovudine treatment can induce mitochondrial multisystem disease, as revealed in our case by myopathy, liver steatosis and lactic acidosis.

IDOVUDINE was the first nucleoside analogue used in the treatment of human immunodeficiency, (HIV) infection (1). It is still widely used in current combination therapies (2). Nucleoside analogues are prodrugs which must be converted into their active, triphosphorylated form. Once incorporated into growing DNA chains, phosphorylated nucleoside analogues act as chain terminators. They inhibit mitochondrial (rot) DNA polymerase gamma and induce mtDNA depletion (3,4). All nucleoside analogues used in the treatment of HIV infection cause mitochondrial toxicity in vitro (5). Didanosine has been associated with fulminant liver failure and microvacuolar steatosis (6,7), sometimes associated with lactic acidosis (7). Hepatic dysfunction and lactic acidosis have also been ob-

served in one patient treated with stavudine (8). Fialuridine, another nucleoside analogue, has induced severe hepatic failure and lactic acidosis associated with mitochondrial myopathy in patients treated for hepatitis B virus infection (9). Its mitochondrial toxicity has been confirmed in vitro (10,1 I). Toxic manifestations due to nucleoside analogue treatment include didanosine-induced pancreatitis and zalcitabine-induced peripheral neuropathy, thought also to be mitochondrial disorders (4). Zidovudine is known to be responsible for a mitochondrial myopathy with ragged-red fibres, myofilamentous abnormalities, lipid droplet accumulation, cytochrome c oxidase deficiency, elevated blood lactate to pyruvate ratios, and mtDNA depletion in muscle (12-17). Lactic acidosis alone (18,19) or associated with hepatic abnormalities including elevated liver enzymes, hepatic failure, hepatomegaly, or steatosis (2022) has also been reported. Such liver damage may also be present without lactic acidosis (23-26). A single re-

Z

Received 27 March; revised 9 July," accepted 5 August 1998

Correspondence: Patrick Chariot, Dtpartement de Pathologie, Htpital Henri Mondor, 94010 Crtteil, France. Tel: 0033 1 4981 2734. Fax: 0033 1 4981 2733. 156

Key words: HIV; Lactic acidosis; Liver; Mitochondria; Mitochondrial DNA; Skeletal muscle; Steatosis; Zidovudine.

Zidovudine-induced mitochondrial disorder

port mentioned the concomitant occurrence of muscular and hepatic changes and lactic acidosis in a patient receiving zidovudine, but muscle and liver tissues were not studied (21). We report here the case of an HIV-infected patient treated with zidovudine, who had liver steatosis, lactic acidosis, and typical features of zidovudine myopathy. M t D N A depletion was shown in liver and muscle. We postulate that this patient presented with a multiorgan mitochondrial disease, probably induced by zidovudine therapy.

choic liver. Blood gas showed lactic acidosis with low bicarbonate level at 17 mmol/1 (n: 22-38). Lactacidaemia was 8 mmol/l (n< 1.6). Zidovudine treatment was stopped. The CK, AST, and ALT levels normalised within 5 days. The lactacidaemia decreased to 6.3 mmol/1 but did not normalise. Acidosis persisted, arterial blood gas pH was 7.34. The patient became confused and had moderate fever (38.5°C). He died 8 days after detection of high blood lactic acid. Post-mortem brain examination showed non-specific reactive gliosis.

Case History

Methods

A 57-year-old man with acquired immunodeficiency syndrome (AIDS) was admitted to hospital for fatigue, weight loss, and increased hepatic enzyme levels. Three years earlier, in February 1993, the patient had tested positive for antibodies against HIV1. The CD4 cell count was 130/mm 3. The anti-HBc antibody test was positive. The anti-HBs antibody and Hbs antigen tests were negative. Testing for anti-HCV antibodies was negative. The patient was clinically asymptomatic. Zidovudine treatment (750 mg per day) and prophylaxis for P. carinii infection with sulfamethoxazole and trimethoprim (SMX/TMP) were given. In May 1994, the creatine kinase (CK) level was normal. In December 1994, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels increased (5- and 4-fold the normal value, respectively). Bilirubin and alkaline phosphatase levels were normal. A mild increase of CK level (2-fold) was noted. The CD4 cell count was 91/mm 3. Zidovudine was stopped and replaced by didanosine (400 mg per day). The ALT and AST levels decreased to 2- and 3 times the normal value within 4 months. Amylase levels increased to 139 U/1 (n< 82). In July 1995, didanosine was stopped and zidovudine was given (750 mg per day). In January 1996, the patient had recurrent diarrhoea, abdominal pain, fatigue, and a moderate weight loss. The AST (6-fold), ALT (8-fold), and gamma glutamyl transferase (3-fold) were elevated. An abdominal computed tomographic (CT) scan was normal. The CD4 cell count was 18/ mm 3. The CK level was moderately elevated (3-fold). The patient's condition worsened in the following weeks. He developed nausea and vomiting, was weak and lost 20% of his weight within 5 months. The CK level increased up to 18-fold. The AST and ALT levels increased up to 8 and 9 times the normal level. Plasma bilirubin, alkaline phosphatase, and albumin levels were normal. Tests for antinuclear antibodies were negative. Prothrombin time was normal at 12.3 s. Abdominal ultrasonography showed an enlarged hypere-

Pathological tissue Liver biopsy was fixed in formalin, embedded in paraffin, and stained with haematoxylin-eosin, picrosirius red for collagen, and Perls' method for iron. A muscle fragment was obtained from the quadriceps at autopsy. It was frozen in liquid nitrogen-cooled isopentane (-160°C) and stored at -80°C. Serial 8-/an-thick cross sections were stained with conventional stains (haematoxylin and eosin, modified Gomori trichrome and oil-red-O) and histochemical reactions for cytochrome c oxidase (complex IV) and succinate dehydrogenase (part of complex II) were performed (27). A liver fragment obtained at autopsy was stained with haematoxylin-eosin and oil-red-O.

rntDNA analysis Quantitative determination of mtDNA was performed by Southern blot analysis. Total DNA was isolated from frozen tissue samples and digested with BamH1. Digested DNA was Southern blotted using a 32p radioactive hybridisation system. Total mtDNA isolated from human placenta (28) and 28S ribosomal cDNA (29) (obtained from Jim Sylvester, New York, USA) were used as probes. Densitometric quantification of the blots, expressed in arbitrary units, was performed using the NIH Image 1.60 software. Signals were corrected for background noise, normalised to the 28S ribosomal cDNA signal of the same lane, and compared to DNA samples analysed on the same blot.

Results Pathological findings Liver biopsy revealed marked, diffuse steatosis, predominantly macrovacuolar, but also microvacuolar (Fig. 1). Portal and periportal fibrosis was mild and contained rare mononuclear inflammatory cells. Hepatocellular necrosis was minimal. Examination of skeletal muscle tissue obtained at autopsy showed marked mitochondrial abnormalities. They consisted of: (a) the presence of ragged-red fibres accounting for 11% of the fibres on trichrome stain (Fig. 2A); (b) an increased succinate dehydrogenase activity in 22% of the fibres (Fig. 2B); and (c) a negative reaction for cytochrome c oxidase in 25% of the fibres. These abnormalities were associated with moderate lipid droplet accumulation and myofilamentous loss. There was no inflammatory infiltration. Examination of liver tissue obtained at autopsy showed massive macro- and microvacuolar steatosis. 157

P. Chariot et al.

skeletal

mtDNA

I i ver

muscle

~,~ ....

nDNA

Fig. 1. Liver biopsy. Marked macrovacuolar steatosis. Rare lipid microvacuoles can be seen (×500).

f--

N

¢O

o

~

~

tt~

I,D

o

o

c

"8

"6

"~

e-

r-

C

~

e-

C

e-

c~

0

0

0

m

0

0

0

Fig. 3. Southern blot analysis'. Total DNA was obtained from skeletal muscle and liver samples and digested by BamH1. Each lane presents the signals obtained from hybridisation of the same membrane with the mtDNA- and nuclear DNA probes. Signals were normalised to the 28S ribosomal cDNA signal of the same lane and compared to DNA samples analysed on the same blot. The proportion of the patient's mtDNA vs nDNA is lower than in controls, which demonstrates mtDNA depletion.

three control liver samples obtained at autopsy (Fig. 3). The amount of the patient's mtDNA in myocardium and kidney was similar to that found in controls (not shown).

Discussion

Fig. 2. Zidovudine myopathy. A." Ragged-red fibres with myofilamentous loss and moderate atrophy (cryostat section, trichrome, ×500). B." Succinate dehydrogenase hyperactivity predominates in atrophic fibres (cryostat section, succinat e dehydrogenase, × 300).

m t D N A analysi~ The amount of the patient's skeletal muscle mtDNA ranged from 20 to 24% (median: 22%) of three control muscle samples (Fig. 3). The amount of the patient's liver mtDNA ranged from 30 to 52% (median: 51°/,,) of 158

This case is remarkable for the occurrence of lactic acidosis associated with liver and muscle abnormalities in a patient treated with zidovudine. The responsibility of zidovudine is strongly suggested by chronological, experimental, histological, and biochemical arguments. Liver and muscle enzyme levels were normal before the beginning of zidovudine therapy and increased while zidovudine was given. Enzyme levels decreased after replacement of zidovudine by didanosine, then markedly increased when zidovudine was reintroduced and normalised a few days after withdrawal of zidovudine. The patient was treated with SMX/TMP until his death. It has been shown that SMX/TMP has no effect on pharmacokinetics of zidovudine in HIV-infected patients (30). No interaction between SMX/TMP and zidovudine has ever been reported and an increased toxicity of zidovudine due to SMX/TMP is unlikely. In experimental models, zidovudine can induce lactate elevation (31) and morphological alterations of muscle (i.e. subsarcolemmal accumulation of mitochondria) (32) and liver (i.e. macro- and microvacuolar steatosis) (33). In our patient, steatosis was macro- as well as microvacuolar. Liver abnormalities induced by zidovudine in animal models include microvacuolar steatosis and

Zidovudine-induced mitochondrial disorder

mitochondrial alterations at electron microscopy (32). Microvacuolar steatosis is the most typical liver abnormality observed in genetically-induced mitochondrial disorders and suggests fl-oxidation defects and respiratory chain abnormalities (34,35). Thus, liver toxicity due to zidovudine is probably related to mitochondrial dysfunction. The existence of lactic acidosis, which traduces impaired redox status (36), further reinforces the hypothesis of a mitochondrial disease in our patient. Lipid droplet accumulation similar to that observed in liver was present in skeletal muscle, supporting a common mechanism of the lesions. Multiorgan involvement is commonly observed in genetically-induced mitochondrial diseases (37), including those with m t D N A depletion, which can affect skeletal muscle, kidney, and liver (38). m t D N A depletion has been found in the skeletal muscle of patients with zidovudine myopathy (17,39), but quantification of m t D N A in other organs has never been reported in HIV-infected patients, either in liver or heart of patients with zidovudine myopathy, or in liver or heart of patients with zidovudine-associated liver or cardiac dysfunction (17,20-24,39,40). In our patient, m t D N A depletion was found in liver and skeletal muscle samples. No depletion was found in kidney and myocardium, and we note that renal failure or clinical symptoms of cardiac dysfunction were absent. It has been proposed that the diagnosis of mitochondrial disorder should be considered in patients with an unexplained association of symptoms involving different organs (41). Zidovudine treatment can induce a mitochondrial multisystem disease, revealed in our case by myopathy, massive hepatic steatosis, and lactic acidosis. Levels of CK, lactate, and liver enzymes should be monitored in patients treated with this compound. Current therapy of HIV-infected patients, which is based on combinations associating several nucleoside analogs and protease inhibitors (2), might enhance drug-related mitochondrial damage. Acknowledgements This study was supported by a grant to P. Chariot from Sidaction (Paris, France). References 1. The Delta Coordinating Committee: Delta: a randomized double-blind controlled trial comparing combinations of zidovudine plus didanosine or zalcitabine with zidovudine alone in HIVinfected individuals. Lancet 1996; 348: 283-91. 2. Hammer S. Advances in an antiretroviral therapy and viral load monitoring. AIDS 1996; 10 (suppl 3): St-11. 3. Yarchoan R, Mitsuya H, Myers CE, Broder S. Clinical pharmacology of 3'-azido-2'3'-dideoxythymidine (zidovudine) and related dideoxynucleosides. N Engl J Med 1989; 321: 726-38.

4. Lewis W, Dalakas MC. Mitochondrial toxicity of antiviral drugs. Nat Med 1995; 1: 417-22. 5. Chen CH, Vazquez-Padua M, Cheng YC. Effect of anti-human immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity. Mol Pharmacol 1991; 39: 625-8. 6. Lai KK, Gang DL, Zawacki JK, Cooley TP. Fulminant hepatic failure associated with 2',3'dideoxyinosine (ddI). Ann Intern Med 1991; 115: 283-4. 7. Bissuel F, Bruneel E Habersetzer F, Chassard D, Cotte L, Chevallier M, et al. Fulminant hepatitis with severe lactate acidosis in HIV-infected patients on didanosine therapy. J Intern Med 1994; 235: 367-72. 8. Lenzo NP, Garas BA, French MA. Hepatic steatosis and lactic acidosis associated with stavudine trearment in an HIV patient: a case report AIDS 1997; 11: 1294-6. 9. McKenzie R, Fried MW, Sallie R, Conjeevaram H, Di Bisceglie AM, Park Y, et al. Hepatic failure and lactic acidosis due to fialuridine (FIAU), an investigational nucleoside analogue for chronic hepatitis B infection. N Engl J Med 1995; 333: 1099-105. 10. Semino-Mora C, Leon-Monzon M, Dalakas MC. Mitochondrial and cellular toxicity induced by fialuridine in human muscle in vitro. Lab Invest 1997; 76: 487-95. 11. Lewis W, Levine ES, Grinuviene B, Tankersley KO, Colacino JM, Sommadossi JP, et al. Fialuridine and its metabolites inhibit DNA polymerase y at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts. Proc Natl Acad Sci USA 1996; 93: 3592-7. 12. Dalakas MC, Ilia I, Pezeshkpour GH, Laukaitis JP, Cohen B, Griffin JL. Mitochondrial myopathy caused by long-term zidovudine therapy. N Engl J Med 1990; 322: 1098-105. 13. Mhiri C, Baudrimont M, Bonne G, Geny C, Degoul F, Marsac C, et al. Zidovudine myopathy: a distinctive disorder associated with mitochondrial dysfunction. Ann Neurol 1991; 29: 606-14. 14. Chariot P, Gherardi R. Partial cytochrome c oxidase deficiency and cytoplasmic bodies in patients with zidovudine myopathy. Neuromusc Disord 1991; 1: 357-63. 15. Tomelleri G, Tonin P, Spadaro M, Tilia G, Orrico D, Barelli A, et al. AZT-induced mitochondrial myopathy. Ital J Neurol Sci 1992; 13: 723-8. 16. Chariot P, Monnet I, Mouchet M, Rohr M, Lefaucheur JP, Dubreuil-Lemaire ML, et al. Blood lactate/pyruvate ratio as a non invasive test for the diagnosis of zidovudine myopathy. Arthritis Rheum 1994; 37: 583-6. 17. Arnaudo E, Dalakas M, Shanske S, Moraes CT, DiMauro S, Schon EA. Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 1991; 337: 508-10. 18. Chattha G, Arieff AL, Cummings C, Tierney LM. Lactic acidosis complicating the acquired immunodeficiency syndrome. Ann Intern Med 1993; 118: 37-9. 19. Maslo C, Jacomet C, Jupas JJ, Lebrette MG, Rozenbaum W. Acidose lactique chez des patients infect6s par le VIH. Presse Med 1994; 23:717. 20. Freiman JP, Helfert KE, Hamrell MR, Stein DS. Hepatomegaly with severe steatosis in HIV-seropositive patients. AIDS 1993; 7: 379-85. 21. Le Bras P, D'Oiron R, Quertainmont Y, Halfon P, Caquet R. Metabolic, hepatic and muscular changes during zidovudine therapy: a drug-induced mitochondrial disease? AIDS 1994; 8: 7167. 22. Olano JP, Borucki M J, Wen JW, Haque AK. Massive hepatic steatosis and lactic acidosis in a patient with AIDS who was receiving zidovudine. Clin Infect Dis 1995; 21: 973-6. 23. Dubin G, Braffman MN. Zidovudine-induced hepatotoxicity. Ann Intern Med 1989; 110: 85-6. 24. Gradon JD, Chapnick EK, Sepkowitz DV. Zidovudine-induced hepatitis. J Intern Med 1992; 231: 317-8. 25. Chen SCA, Barker SM, Mitchell DH, Stevens SMB, O'Neill P,

159

P. Chariot et al.

26.

27.

28. 29.

30.

31.

32.

33.

34.

Cunningham AL. Concurrent zidovudine-induced myopathy and hepatotoxicity in patients treated for HIV infection. Pathology 1992; 24:109-11. Fortgang I, Belitsos P, Chaisson R, Moore R. Hepatomegaly and steatosis in HIV-infected patients receiving nucleoside analog antiretroviral therapy. Am J Gastroenterol 1995; 90: 1433-6. Chariot R Monnet I, Gherardi R. Cytochrome c oxidase reaction improves histopathological assessment of zidovudine myopathy. Ann Neurol 1993; 34:561 5. Drouin J. Cloning of human mitochondrial D N A in Escherichia coil. J Mol Biol 1980; 140: 15-34. Gonzalez IL, Gorski JL, Campen T J, Dorney D J, Erickson JM, Sylvester JE, et al. Variation among human 28S ribosomal RNA genes. Proc Natl Acad Sci USA 1985; 82: 7666-70. Canas E, Pachon J, Garcia-Pesquena E Castillo JR, Villana P, Cisneros JM, et al. Absence of effect of trimethoprim-sulfamethoxazole on pharmacokinetics of zidovudine in patients infected with human immunodeficiency virus. Antimicrob Agents Chemother 1996; 40: 230-3. Lamperth L, Dalakas MC, Dagani E Anderson J, Ferrari R. Abnormal skeletal and cardiac muscle mitochondria induced by zidovudine (AZT) in human muscle in vitro and in an animal model. Lab Invest 1991; 65: 742-51. Corcuera T, Alonso M J, Picazo A, Gomez F, Roldan M, Abad M, et al. Hepatic morphological alterations induced by zidovudine in an experimental model. Pathol Res Pract 1996; 192: 182-7. Benbrik E, Chariot P, Bonavaud S, Ammi-Said M, Rey C, Gherardi R, et al. Cellular and mitochondrial toxicity of zidovudine (AZT), didanosine (ddl), and zalcitabine (ddC) on cultured human muscle cells. J Neurol Sci 1997; 149: 19-25. Bioulac-Sage P, Parrot-Roulaud F, Mazat JR Lamireau T, Coquet

160

35.

36.

37.

38.

39.

40.

41.

M, Sandier B, et al. Fatal neonatal liver failure and mitochondrial cytopathy (oxidative phosphorylation deficiency): a light and electron microscopic study of the liver. Hepatology 1993; 18: 839-45. Fromenty B, Berson A, Pessayre D. Microvesicular steatosis and steatohepatitis: role of mitochondrial dysfunction and lipid peroxidation. J Hepatol 1997; 26 (suppl 1): 13-22. Poggi-Travert E Martin D, Billette de Villemeur T, Bonnefont JP, Vassault A, Rabier D, et al. Metabolic intermediates in lactic acidosis: compounds, samples and interpretation. J Inter Metab Dis 1996; 19:478 88. Hammans SR, Morgan-Hughes JA. Mitochondrial myopathies: clinical features, investigation, treatment and genetic counselling. In: Di Mauro S, Schapira AHV. editors. Mitochondriat Disorders in Neurology. Oxford: Butterworth-Heinemann; 1994. pp. 4974. Moraes C, Shanske SE, Tritschler H J, Aprille JR, Andreetta E Bonilla E, et al. MtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases. Am J Hum Genet 1991: 48: 492-501. Casademont J, Barrientos A, Gruau JM, Pedrol E, Estivilli X, Urbano-Marquez A, et al. The effects of zidovudine on skeletal muscle mt-DNA in HIV-1 infected patients with mild or no muscle dysfunction. Brain 1996; 119:1357 64. Herskowitz A, Sharon B, Baughman K, Schulman S, Bartlett J. Cardiomyopathy associated with antiretroviral therapy in patients with HIV infection: a report of six cases. Ann Intern Med 1992; 116:311 3. Munnich A, RStig A, Chr6tien D, Cormier V, Bourgeron T, Bonnefont JP, et al. Clinical presentation of mitochondrial disorders in childhood. J Inher Metab Dis 1996; 19:521 7.