| The present research was conducted to explore the potential mitochondrial toxicities and their severities of intravenously administered metacavir, a nucleoside analog,in rhesus monkeys, and to provide the clinical references for safe use of the drug.Totally 27 rhesus monkeys were randomly divided into 5 groups:metacavir 120 mg/kg group(6 monkeys), metacavir 40 mg/kg group(6 monkeys),metacavir 13 mg/kg group(6 monkeys), zidovudine(AZT) 50 mg/kg group as the positive control drug(3 monkeys),and blank control group(6 monkeys, intravenously administered with normal saline).Animals were killed after the completion of a 3-month dosing trial or further observed in a 4-week recovery phase. Drugs/placebo was given 6 times every week for consecutively three months, and drugs was oral administered in AZT group and intravenously administered in other groups.In the metacavir groups and blank control group,4 animals were killed 3 months after the start of dosing, and the remaining 2 animals in each group were used for observation during the 4-week recovery phase. In AZT 50 mg/kg group, all animals were killed 3 months after the start of dosing. Pathological changes in tissues were observed under light microscope. Changes of the ultrastructure of mitochondria in liver, kidney, skeletal muscles, and cardiac muscles were observed under transmission electron microscope(TEM). Changes of the activities of mitochondrial respiratory chain complexes I-IV and mitochondrial DNA were also determined, respectively use spectrophotometry and quantitative PCR.The results indicated that:(1)Clinical observation:nausea, vomiting and decreased appetite observed in AZT group;No obvious clinical symptoms occurred in Metacavir groups, either 3 months administration or recovery phase. (2)Histopathology observation:Rhesus monkeys in Metacavir 120mg/kg group(4/4) in 3 months administration observed pathological changes, involve of liver, kidney, small intestine and stomach. Liver and kidney(4/4),stomach(3/4) and small intestine(1/4) in Metacavir 40mg/kg group rhesus monkeys in 3-month administration occurred pathological changes.Other tissues of rhesus monkeys in Metacavir 120mg/kg group and Metacavir 40mg/kg group unobserved pathological changes. Liver and kidney(3/3) in AZT 50mg/kg group rhesus monkeys observed pathological changes. Tissues of Metacavir 13mg/kg group and blank control group in 3-month administration observed pathological changes. None pathological changes occurred in recovery phase in all groups. (3)Results of Ultramicro-structure of mitochondria with-transmission electron microscope (TEM):After the first 3-month dosing trial, all the killed animals in the metacavir 40 mg/kg group and bland control group, no obvious drug-related mitochondrial injuries in skeletal muscle, cardiac muscle, kidney, or liver cells were noted. Among all the killed animals (4/4) in the metacavir 120 mg/kg group, no obvious mitochondrial injuries in cardiac muscle or kidney were noted, but the loss of part of the liver glycogen particles was found. The dosing-relevant broken cristae and sparse cristae were found in skeletal muscle cells, while their morphologies and sizes had no remarkable changes in the metacavir 120 mg/kg group.No obvious mitochondrial were injuries observed in above two groups.Compared with the blank control group, among all the killed animals (3/3)in AZT 50 mg/kg group, the mitochondria in liver, kidney, skeletal muscle, and cardiac muscle experienced relatively obvious injuries, although their severities varied. (4) Results of the activities of respiratory chain complexesâ… -â…£:â‘ complexâ… :After the first 3-month dosing, compared with the blank control group, the activities of mitochondria in liver, kidney, skeletal muscle, arid cardiac muscle in AZT group were decreased obviously about 50%(P<0.01),also the activities of mitochondria decreased significantly (P<0.05).During the recovery phase, no obvious diversities were noted between metacavir or AZT group and blank control group.â‘¡complexâ…¡:After the first 3-month dosing, compared with the blank control group, the activities of mitochondria in skeletal muscle, and cardiac muscle in AZT group were decreased obviously (P<0.01),also activities in liver decreased significantly(P<0.05),while the activitie in liver was tend to decrease, but no significant difference. The activities of mitochondria in skeletal muscle, and cardiac muscle in Metacavir 120mg/kg group were decreased obviously (P<0.05),During the recovery phase, no obvious diversities were noted between metacavir or AZT group and blank control group.â‘¢complexâ…¢: After the first 3-month dosing, compared with the blank control group, the activities of mitochondria in liver, kidney, and skeletal muscle in AZT group were decreased obviously (P<0.01),also activities in cardiac muscle decreased significantly (P<0.05), no obvious diversities were noted between metacavir group and blank control group. During the recovery phase, no obvious diversities were noted between metacavir or AZT group and blank control group.â‘£complex IV:After the first 3-month dosing, compared with the blank control group, the activities of mitochondria in mitochondria in AZT group were decreased obviously (P<0.01),also activities in kidney and skeletal muscle decreased significantly (P<0.05),no obvious diversities were noted between metacavir group and blank control group.During the recovery phase, no obvious diversities were noted between metacavir or AZT group and.blank control group.(5)Effects on mtDNA contents:As shown by QPCR in the quantitative determination of mtDNA (Cytochrome b gene) and nuclear nDNA (Actin gene), the cytochrome b/actin ratio varied among different rhesus monkeys.After 3 months of experiment, compared with the blank control group, in AZT 50 mg/kg group, mtDNA content signifcantly decreased in skeletal muscle, cardiac muscle, and liver by 70% or higher (P<0.01)and also signifcantly decreased in kidney (P<0.05),However, in the metacavir 120 mg/kg group, the mtDNA contents significantly decreased in skeletal muscle (P<0.05),and liver (P<0.05),and cardiac muscle (P<0.01);but they were all higher than those in AZT 50 mg/kg group.On week 4 in the recovery phase, the mtDNA contents in all tissues in the metacavir 120 mg/kg group showed rising trends.Conclusion:(1)In AZT 50mg/kg group, there were evident ultramicro-structure changes and dysfunction in mitochondria of liver, kidney, skeletal muscle, and cardiac muscle after 3-month dosing trial.Meanwhile decreased complexes activities and mtDNA content(copies).(2) Liver, skeletal muscle, and cardiac muscle shown varies mitochondrial damage, and the degree is:skeletal muscle>liver, cardiac muscle, weak damage on renal mitochondrial.There were recovery trend from toxicity indicators after 4-week withdrawal.Weaker mitochondrial damage compared metacavir 120 mg/kg dosage to AZT 50mg/kg dosage. (3)No obvious mitochondrial injuries in tissues were noted in Metacavir 40mg/kg group, and mitochondrial function unchanged, which can be used as a reference frame to design clinical dose for human use and major indicators.(4)Metacavir has not a high risk for potential mitochondrial-related effects in rhesus monkeys, and all the injuries in tissues caused by Metacavir were reversible. |
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