Immunohistochemical analysis of expressions of hepatic cytochrome P450 in F344 rats following oral treatment with kava extract

Abstract

Kava (Piper methysticum), used for relaxation and pain relief, has been one of the leading dietary supplements and several reports linking hepatic functional disturbances and liver failure to kava have resulted in a ban on sales in Europe and Canada and the issuance of warnings by the US FDA. The National Toxicology Program conducted 14-week rat studies to characterize the toxicology of kava exposure in Fischer 344 rats [National Toxicity Program. 90 day gavage toxicity studies of KAVA KAVA EXTRACT in Fischer rats and B6C3F1 mice. Research Triangle Park, NC; 2005a; National Toxicity Program. Testing status of agents at NTP (KAVA KAVA EXTRACT M990058). Research Triangle Park, NC; 2005b. (http://ntp.niehs.nih.gov/index.cfm?objectid=071516E-C6E1-7AAA-C90C751E23D14C1B)]. Groups of 10 male and 10 female rats were administered kava extract by gavage at 0, 0.125, 0.25, 0.5, 1.0, and 2.0 g/kg/day. Increased gamma-glutamyl-transpeptidase (GGT) activities were observed in the 2.0 g/kg males and 1.0 and 2.0 g/kg females, as well as increased serum cholesterol levels in males and females at 0.5 g/kg and higher. Increases in incidence and severity of hepatocellular hypertrophy (HP) were noted in males at 1.0 g/kg and females at 0.5 g/kg and higher, as well as increased liver weights. Immunohistochemical analyses of the expression of cytochrome-P450 (CYP) enzymes in liver of the control and 1.0- and 2.0-g/kg-treated groups indicated decreased expression of CYP2D1 (human CYP2D6 homolog) in 2.0 g/kg females and increased expression of CYP1A2, 2B1, and 3A1 in 1.0 and 2.0 g/kg groups of both sexes. The no observed adverse effect levels were decided as 0.25 g/kg in both genders, based on neurotoxic effects, increases in GGT, cholesterol, liver weight, and HP and decreases in body weight. Kava-induced hepatic functional changes in the F344 rat might be relevant to human clinical cases of hepatotoxicity following exposure.

Figs. 1 and 2

1. Centrilobular area, control female rat. Note relatively smaller size of hepatocytes with cytoplasmic basophilic stippling by comparing with liver from 14-week-, 2.0 g/kg-treated rat. C: central vein. G: Glison’s sheath. H&E. Bar = 50 μm. 2. Mild (grade 2) hepatocytic hypertrophy in female rat treated with 2.0 g/kg kava extract by gavage for 14 weeks. Centrilobular hepatocytes contain more homogeneous eosinophic cytoplasm. C: central vein. G: Glison’s sheath. H&E. Bar = 50 μm.

Figs. 3–6

3. Strong CYP2D1 expression (intensity: grade 3) in centrilobular area, control female rat; CYP2D1 detected diffusely in cytoplasm of hepatocytes of controls. C: central vein. G: Glison’s sheath. Bar = 50 μm. 4. Higher magnification of Fig. 3. C: central vein. Bar = 50 μm. 5. Moderate expression (intensity: grade 2) of CYP2D1 in centrilobular area of hepatocytes showing mild hypertrophic changes by H&E staining, female rat treated with 2.0 g/kg kava extract by gavage for 14 weeks. C: central vein. G: Glison’s sheath. Bar = 50 μm. 6. Higher magnification of Fig. 5. C: central vein. Bar = 50 μm.

Figs. 7–10

7. Weak expression (relative area: grade 1) of CYP2B1, centrilobular area only, control female rat. CYP2B1 protein was detected only in cytoplasm of centrilobular hepatocytes in control rats. C: central vein. G: Glison’s sheath. Bar = 50 μm. 8. Higher magnification of Fig. 7. C: central vein. Bar = 50 μm. 9. Strong expansive expression (relative area: grade 4) of CYP2B1 in almost all of lobular area, detected in hepatocytes showing hypertrophic changes by H&E staining, female rat treated with 2.0 g/kg of kava extract by gavage for 14 weeks. C: central vein. G: Glison’s sheath. Bar = 50 μm. 10. Higher magnification of Fig. 9. C: central vein. Bar = 50 μm.

Figs. 11–14

11. Weak expression (relative area: grade 1) of CYP3A1 only in centrilobular area, detected locally in cytoplasm of hepatocytes around central vein, control female rat. C: central vein. G: Glison’s sheath. Bar = 50 μm. 12. Higher magnification of Fig. 11. C: central vein. Bar = 50 μm. 13. Strong expression (relative area: grade 4) of CYP3A1 in almost all of centrilobular area detected by H&E in cytoplasm of hepatocytes showing hypertrophic changes, female rat treated with 2.0 g/kg of kava extract by gavage for 14 weeks. C: central vein. Bar = 50 μm. 14. Higher magnification of Fig. 13. C: central vein. Bar = 50 μm.

Figs. 15–18

15. Weak expression (relative area: grade 1) of CYP1A2 only in centrilobular area, control female rat. CYP1A2 protein was detected locally in cytoplasm of hepatocytes around the central vein. C: central vein. G: Glison’s sheath. Bar = 50 μm. 16. Higher magnification of Fig. 15. C: central vein. Bar = 50 μm. 17. Strong expression (relative area: grade 3) of CYP1A2, centrilobular area, female rat treated with 2.0 g/kg of kava extract by gavage for 14 weeks. CYP1A2 detected in the cytoplasm of hepatocytes showing hypertrophic changes by H&E staining. C: central vein. G: Glison’s sheath. Bar = 50 μm. 18. Higher magnification of Fig. 17. C: central vein. Bar = 50 μm.