Palmitoyl-CoA, oxidation

Hepatocytes isolated from male Wistar rats (180-250 g) were treated with 0.2 mM mono(2-ethylhexyl) phthalate or 1 mM 2-ethylhexanol for 48 h (Gray et al., 1982). Both di(2-ethylhexyl) phthalate metabolites increased carnitine acetyltransferase activity about nine-fold. In studies with hepatocytes from male Sprague-Dawley rats (180-220 g), treatment with 0.2 mM mono(2-ethylhexyl) phthalate and 1.0 mM 2-ethylhexanol for 48 h resulted in induction of carnitine acetyltransferase activity about 15-fold and six-fold, respectively (Gray et al., 1983). Mono(2-ethylhexyl) phthalate was also shown to induce cyanide-insensitive palmitoyl-CoA oxidation and, by ultra-structural examination, to increase numbers of peroxisomes. Hepatocytes were isolated from Wistar-derived rats (180-220 g) and treated for 72 h with 0-0.5 mM mono(2-ethylhexyl) phthalate and some mono(2-ethylhexyl) phthalate metabolites (Mitchell etal., 1985). Treatment with mono(2-ethylhexyl) phthalate and metabolites VI and IX (see Figure 1) resulted in a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation. In addition, 0-0.5 mM mono(2-ethylhexyl) phthalate and 0-1.0 mM metabolite VI produced concentration-dependent increases in lauric acid hydroxylation. Treatment with metabolites I and V resulted in only small effects on the enzymatic markers of peroxisome proliferation. In another study with hepatocytes from Wistar-derived rats (180-220 g), metabolite VI was shown by subjective ultrastructural examination to cause proliferation of peroxisomes (Elcombe Mitchell, 1986). 

Primary hepatocyte cultures may also be employed to study species differences in hepatic peroxisome proliferation (lARC, 1995 Doull et al, 1999). Hepatocytes were isolated from male Sprague-Dawley rats (180-220 g), male Syrian hamsters (70-80 g) and male Dunkin-Hartley guinea-pigs (400-450 g). Treatment with 20-200 0,M mono(2-ethylhexyl) phthalate for 70 h caused strong induction of cyanide-insensitive palmitoyl-CoA oxidation activity in rat hepatocytes (up to 600% of control levels), while no marked effect was observed in Syrian hamster (up to 120% of control) or guinea-pig (down to 80% of control) hepatocytes (Lake et al., 1986). 

Hepatocytes were isolated from male Wistar-derived rats (180-220 g) and male Alderley Park guinea-pigs (400-500 g) and treated with 0-0.5 mM mono(2-ethyl-hexyl) phthalate or metabolite IX for 72 h (Mitchell et al., 1985).While both caused concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat hepatocytes, no such effect was observed in guinea-pig hepatocytes. 

Primary hepatocyte cultures may be employed to study species differences in hepatic peroxisome proliferation (lARC, 1995). The effects of di(2-ethylhexyl) adipate and its metabolites in cultured hepatocytes from rats, mice, guinea-pigs and marmosets have been studied (Cornu et al., 1992). In hepatocytes from each species, the parent compound di(2-ethylhexyl) adipate had no effect on peroxisomal cyanide-insensitive palmitoyl-CoA oxidation activity. However, in rat and mouse hepatocytes, the metabolites mono(2-ethylhexyl) adipate, 2-ethylhexanol, 2-ethylhexanoic acid and 2-ethyl-5-hydroxyhexanoic acid at concentrations < 1 mM induced peroxisomal palmitoyl-CoA oxidation. No induction of peroxisomal palmitoyl-CoA oxidation was seen at concentrations < 1 mM for mono(2-ethylhexyl) adipate or < 2 mM for 2-ethylhexanol, 2-ethylhexanoic acid and 2-ethyl-5-hydroxyhexanoic acid in guinea-pig or marmoset hepatocytes (2-ethylhexanol was evaluated only at < 1 mM in marmoset hepatocytes). 

Cultured hepatocytes from non-human primates (marmosets and macaques) and humans have been similarly unresponsive to a variety of peroxisome proliferators (reviewed in Doull et al., 1999). No evaluation of peroxisome proliferation in human hepatocytes treated with di(2-ethylhexyl) adipate metabolites in vitro has been published. The lack of peroxisome proliferation in hepatocytes from marmosets suggests that human hepatocytes also would be unresponsive (Cornu et al., 1992). These negative results were significant in that the same metabolites induced typical induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation activity in rat and mouse hepatocytes. 

The hepatic effects of cinnamyl anthranilate were evaluated in male CD 1 mice and male Fischer 344 rats treated by intraperitoneal injection for three consecutive days (Viswalingam Caldwell, 1997). At doses of 100 and 1000 mg/kg bw per day, relative liver weights of mice increased by 22% and 50%, respectively, 24 h after the final dose and peroxisomal (cyanide-insensitive) palmitoyl-coenzyme A (CoA) oxidation activity increased fivefold at both levels. Microsomal lauric acid 11- and 12-hydroxylase activity (CYP4A) was increased 15-fold at 100 mg/kg bw per day and 17-fold at 1000 mg/kg bw per day. Limited evaluation indicated that cirmamyl anthmilate increased the size and number of peroxisomes in electron micrographs of hepatocytes of treated mice. In rats, relative liver weights and peroxisomal palmitoyl-CoA oxidation activity were significantly increased only at 1000 mg/kg bw per day (22% and twofold, respectively). 

In a separate experiment, groups of male CDl mice were given intraperitoneal injections of 0-200 mg/kg bw ciimamyl anthranilate daily for three days. At doses of 20 mg/kg bw and above, there were dose-dependent increases in relative liver weight, total cytochrome P450, and cyanide-insensitive palmitoyl-CoA oxidation. The hepatic effects of cinnamyl anthranilate are apparently due to the intact ester, since neither its expected metabolites alone nor an equimolar mixture of the hydrolysis products, cinnamyl alcohol and anthranilic acid, had a significant effect on the weight or marker enzyme content of mouse liver (Viswalingam Caldwell, 1997). 

The complete oxidation of 1 mole of palmitic acid to C02 and H20 produces NADH and FADH2 by /3-oxidation and by citric acid cycle activity. ATP synthesis is coupled to the oxidation of NADH and FADH2 produced in these processes (Chap. 14). Per mole of palmitoyl-CoA oxidized, 131 moles of ATP are synthesized. But. two high energy bonds of ATP are used in the formation of palmitoyl-CoA, so the net ATP production is 129. 

Fig. 2. Relative Peroxisome Proliferative Activity of Chlorinated Phenoxyacetic Acids and Related Compounds in Cultured Rat Hepatocytes. ( R.P. = Relative potency for induction of palmitoyl CoA oxidation. Data summarized from Lewis et al. [20])...

In a study of dry cleaning workers in China, urinary metabolite levels (total trichloro compounds) were reduced when workers were exposed to mixtures of tetrachloroethylene and trichloroethylene as opposed to trichloroethylene alone (Seiji et al. 1989). The effect on the trichloroethylene metabolite, trichloroethanol was greatest, with little effect on TCA, a metabolite of both trichloroethylene and tetrachloroethylene. The study authors indicated that because of the smaller amount of tetrachloroethylene metabolized, it was not possible to determine if trichloroethylene suppressed the metabolism of tetrachloroethylene. Concurrent administration of tetrachloroethylene and trichloroethylene to mice did not result in additive or synergistic effects in induction of hepatic peroxisomal proliferation as measured by cyanide-insensitive palmitoyl CoA oxidation activity (Goldsworthy and Popp 1987). This may be related to preferential metabolism of trichloroethylene at the dose levels used. 

Krauss, S., Lascelles, C.V., Zammit, V.A. Quant, PA. ( 9%)Biochem. J. 319,427 33. Fluxeontrol exerted by overt carnitine palmitoyltransferase over palmitoyl-CoA oxidation and ketogenesis is lower in suckling than in adult rats. 

Thomas, J., Debeer, L.J., De Schepper, PJ. Mannaerts, G.P. (19S0) Biochem. J. 190,485-94. Eactors influencing palmitoyl-CoA oxidation by rat liver peroxisomal fractions. Substrate concentration,... 

In the second trial conducted by Murata et al. (1997), varying amounts of dietary TAG were replaced by DAG while the dietary fatty acid content was maintained at 9.39 g/100 g diet. After 21 days on the new diets, significant reductions in serum and liver TAG levels were found in the groups of rats fed diets in which DAG supplied more than 6.58 gfatty acids/100 g diet. Reductions in the activities of enzymes involved in fatty acid synthesis and increases in palmitoyl-CoA oxidation rates by both mitochondrial and peroxisomal pathways were also apparent when DAG replaced TAG in diets to supply more than 6.58 g fatty acid/100 g diet. Increasing dietary levels of DAG progressively increased the activities of enzymes involved in the P-oxidation pathway in the liver, including carnitine palmitoyltransferase (EC 2.3.1.21), acyl-CoA... 

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