Chronic Phenobarbital-Induced Mitochondrial Pleomorphism in the Rat Liver

Abstract

Posttreatment effects of phenobarbital (30 mg/kg I.P. per day for 5 days) on liver mitochondrial TEM ultra-structure in adult rats were studied. Liver mitochondria population samples in treated rats exhibited pleomorphic morphological dumbbell shapes (7%), U-type (crescent) shapes (4%) and O-type (ring-like) shapes (4%). This pathology was not observed in vehicle-treated animals. Phenobarbital is a drug that induces within the rat liver increased protein synthesis in the mitochondria and mitochondrial morphological shape changes.

Phenobarbital is an anesthetic barbiturate and exerts its CNS effects by interacting with GABA _A receptors. Barbiturates have the potential to cause liver toxicity, such as structural changes to hepatocyte subcellular organelles and induce changes in liver enzymes (Venditti et al., 1998; Gonzales et al., 2005; Sonmez et al., 2006). For example, in experimental rats the chronic response to phenobarbital on liver content is an increase in microsomal and mitochondrial proteins (Venditti et al., 1998). On the other hand, treatment phenobarbital does not induce a significant increase in mitochondrial fraction (Venditti et al., 1998). These combined observations, suggest, that while there is an increase in mitochondrial protein synthesis per se, there may not be a corresponding increase in mitochondrial number. Whether this observation can be explained for example, by an increase in mitochondrial size or other ultra-structural changes has not been previously considered. Thus the aim of the present study is to examine via transmission electron microscopy (TEM) liver mitochondria ultra-structure so as to better characterize responses to the administration of phenobarbital.

Animal ethics were approved by the National University of Singapore and compliance was according to the Singaporean National Advisory Committee for Laboratory Animal Research. Ten adult male Wistar rats, weighing between 450 g and 500 g were used for the present experiments. Five rats were treated with daily injections of Sodium-Phenobarbital (dissolved in 0.9% NaCl) at a dose of 30 mg/kg intraperitoneally for 5 days. Vehicle controls had equivalent volumes of 0.9% NaCl injected. Following sacrifice, 5 days after the last sodium-phenobarbital injection, the liver was quickly removed and fixed in 10 ml of 2.5% gluteraldehyde in 0.1 M cacodylate buffer (pH 7.4) and then processed for TEM as previously described (McLachlan et al., 2007). The classification scheme provided by Ghadially (1997) was used to describe observed mitochondrial morphological shape changes.

Under TEM, liver mitochondria from all vehicle-treated rats had a typical liver homogenous mitochondrial population with classical ultrastructure of hepatic mitochondria containing few cristae and dense matrix (Figure 1A). Whereas in the Phenobarbital treated rats there were numerous alterations of mitochondrial morphology encountered. For example, Figure 1B depicts normal shaped mitochondria intermixed with 3 abnormal shaped mitochondria: (1) stretched or elongated about their axis, with rounded ends that were “dumbbell”; (2) curved such that they represented the letter “U” (3) had ring-like appearance like the letter “O” (Ghadially, 1997). Dumbbell, “U” and “O” shaped forms each occupied 4–7% of the total mitochondrial population following Phenobarbital treatment (Figure 1C).

The stretched center (axis) of these “dumbbell” mitochondria had well-developed parallel cristae and may be the result of opposite sides of the mitochondria coming within close proximity to one another. Dumbbell mitochondria have been observed previously within the liver of the rat following the administration of subchronic toxic doses of 3,3’,4,4’-tetrachlorobiphenyl (PCB) (MacLellan et al., 1994) and in male hamsters at 220 days after experimental infection with the liver fluke Opisthorchis viverrini (Adam et al., 1993). Some of these abnormal mitochondria ultrastructural forms that we have observed in our treated rats have also been reported in yeast (Keyhani, 1987).

It has been reported that morphological ring forms of mitochondria can be found in bone marrow aspirates, from patients treated with large doses of chloramphenicol (Skinnider and Ghadially, 1976). The drug chloramphenicol is an inhibitor of protein synthesis and it has been suggested that mitochondrial ring forms may be associated with this protein synthesis inhibition (Skinnider and Ghadially, 1976). On the other hand in our studies we also observed mitochondrial ring forms following treatment with phenobarbital. Interestingly, phenobarbital has the opposite effects to chloramphenicol, where it has been reported to increase protein mitochondrial synthesis in the liver (Venditti et al., 1998). It is difficult to explain these observations for ring type mitochondrial shapes for 2 drugs that have opposite effects, that is, to increase protein synthesis or cause inhibition. A possible explanation may be rebound increases in protein synthesis, above baseline levels, following pharmacological clearance of chloramphenicol (Raw and Rockwell, 1979).

Thus, posttreatment effects of phenobarbital on rat liver can be observed under TEM, where elongated atypical liver mitochondria in rats are capable of forming dumbbell, U- and O-type shapes. Phenobarbital increases mitochondrial protein synthesis and is associated with complex mitochondrial shapes, although a physiological casual link remains to be determined for our observed association.