Ijaz A. Qureshi

Butyrate mediates Caco-2 cell apoptosis via up-regulation of pro-apoptotic BAK and inducing caspase-3 mediated cleavage of poly-(ADP-ribose) polymerase (PARP).

Butyrate exerts potent anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. Using the Caco-2 cell line, a well established model of colon cancer cells, our data show that butyrate induced apoptosis (maximum 79%) is mediated via activation of the caspase-cascade. A key event was the proteolytic activation of caspase-3, triggering degradation of poly-(ADP-ribose) polymerase (PARP). Inactivation of caspase-3 with the tetrapeptide zDEVD-FMK completely inhibited the apoptotic response to butyrate. In parallel, butyrate potently up-regulated the expression of the pro-apoptotic protein bak, without changing Caco-2 cell bcl-2 expression. Butyrate-induced Caco-2 cell apoptosis was completely blocked by the addition of cycloheximide, indicating the necessity of protein synthesis. However, when this inhibitor was added at a time point where bak expression was already enhanced (12–16 h after butyrate stimulation), it failed to protect Caco-2 cells against apoptosis. Taken together, these data provide evidence that the molecular events involved in butyrate induced colon cancer cell apoptosis include the caspase-cascade and the mitochondrial bcl-pathway.

Ornithine transcarbamylase deficiency in mutant mice I. Studies on the characterization of enzyme defect and suitability as animal model of human disease.

Summary: Eighty-four young mice born from matings of four sparse-fur (spf)/+ females and two 22A +/Y males, were classified according to spf phenotype for males and orotic acid excretion and ornithine transcarbamylase (OTC) activity for females. The OTC activity of 15.2 ± 1.32 μmole citrulline/mg protein/hr shown by hemizygous affected males was 13% of that of normal males. Heterozygous females showed a much wider variation with a mean activity of 56% of normals.Apparent Km and Vmax of the liver OTC in respect of carbamyl phosphate (CP) and ornithine (ORN) were measured for the hemizygous affected males, heterozygous females, and normal males and females. Km and Vmax (CP) for hemizygous affected males were significantly lower than normal males, normal females, and heterozygous females. Mean Km and Vmax (CP) values of heterozygous females were also significantly different from normal groups and hemizygous affected males. There were no significant differences for the values of Km (ORN) among various groups. However, the Vmax (ORN) of hemizygous males was significantly lower than the other three groups. Vmax (ORN) of the heterozygous females was also lower than the normal groups.Mean value of 18.38 ± 5.13 μmole orotic acid/mg creatinine, excreted by spf/Y males, was significantly higher than all other groups. The average excretion of 7.38 ± 5.63 μmole by heterozygous females was also significantly higher than normal males (0.72 ± 0.23 μmole) and females (0.54 ± 0.27 μmole). The high orotic acid excretion by mutant mice underlines the basic similarity of these animals to the human counterpart.There was no significant difference in the excretion of urinary urea between normal males and affected hemizygous males, or between normal females and heterozygous females.Speculation: Lower Km for carbamyl phosphate might be a characteristic of OTC from mutant mice with spf gene, which could be detected by urinary orotic acid measurement. These mice would prove to be useful animal models for studying nutritional and therapeutic measures to alleviate the consequences of OTC deficiency in children. Characterization of the defect of a mutant enzyme in an animal model should lead to greater understanding of the human disease counterpart. These studies would also be helpful in investigation of other inherited hyperammonemic syndromes.

Effects of congenital hyperammonemia on the cerebral and hepatic levels of the intermediates of energy metabolism in spf mice

Sparse-fur (spf) mutant mice with X-linked ornithine transcarbamylase (OTC) deficiency were examined for hyperammonemia and its effect on energy metabolism. We compared the levels of ammonia, glutamine, glutamate and some of the intermediates of energy metabolism in the brain and liver of spf mice with those of control mice. In spf mice we observed significant increases in ammonia, glutamine, alpha-ketoglutarate and glucose with a significant decrease in ATP, glutamate and pyruvate in both brain and liver. The redox states of the brain and liver were also altered in spf mice. The results suggest that many of the metabolic alterations seen in spf mice could be due to the elevated ammonia levels. The spf mouse may, therefore, be an ideal model for the study of the neurotoxic effects of ammonia in chronic hyperammonemic syndromes.

The biochemistry and toxicology of benzoic acid metabolism and its relationship to the elimination of waste nitrogen

Detoxification of sodium benzoate by elimination as a conjugate with glycine, a nonessential amino acid, provides a pathway for the disposal of waste nitrogen. Since 1979, sodium benzoate has been widely used in the therapeutic regimen to combat ammonia toxicity in patients born with genetic defects in the urea cycle. Although the clinical use of benzoate is associated with improved outcome, the search for biochemical evidence in support of the rationale for benzoate therapy has produced conflicting results. This review begins with an historical account leading to elucidation of the biochemistry of benzoate detoxification and early work indicating the potential utility of the pathway for elimination of waste nitrogen. An introduction to contemporary efforts at employing benzoate to treat hyperammonemia is followed by a detailed review of studies on benzoate metabolism and resultant toxic interactions with other major metabolic pathways. With this background, the several metabolic routes by which benzoate is thought to promote the disposal of waste nitrogen are then examined, followed by a consideration of alternative mechanisms by which benzoate might combat ammonia toxicity.

Progressive decrease of cerebral cytochrome C oxidase activity in sparse-fur mice : role of acetyl-L-carnitine in restoring the ammonia-induced cerebral energy depletion

Sparse-fur (spf) mice with a deficiency of hepatic ornithine transcarbamylase (OTC) are congenitally hyperammonemic, showing elevated cerebral ammonia and glutamine and depleted levels of energy metabolites. This mouse disorder is akin to the human OTC deficiency, in which neuronal loss and Alzheimers type II astrocytosis is reported. Reduced cytochrome C oxidase (COX) activity is characteristic of neurodegeneration in Alzheimers type disorders. We have studied the causal relationship between cerebral COX activity and energy depletion in spf mice. Our results indicate a progressive decrease in the COX activity in various brain regions in spf mice, up to 40 weeks of age, which severely effected the cerebral levels of various energy metabolites. A quantitative estimation of cerebral COX subunit I mRNA also showed a tendency to decrease in spf mice. Short-term acetyl L-carnitine (ALCAR) treatment restored these abnormalities. Our study points out that: (a) ammonia-induced alterations in the cerebral reducing equivalents could cause a decrease in COX activity and its mRNA expression, and (b) ALCAR administration could normalize the cerebral energy metabolism and induce COX mRNA expression and activity.

Hereditary and acquired diseases of acyl-coenzyme A metabolism.

Coenzyme A (CoA) sequestration, toxicity or redistribution (CASTOR) is predicted to occur in many hereditary and acquired conditions in which the degradation of organic acyl esters of CoA is impaired. The resulting accumulation of CoA esters and reduction of acetyl-CoA and free CoA (CoASH) will then trigger a cascade of reactions leading to clinical disease. Most conditions detected by expanded neonatal screening are CASTOR diseases. We review acyl-CoA metabolism, including CoASH synthesis, transesterification of acyl-CoAs to glycine, glutamate or l-carnitine and hydrolysis of CoA esters. Because acyl-CoAs do not cross biological membranes, their main toxicity is intracellular, primarily within mitochondria. Treatment measures directed towards removal of circulating metabolites do not address this central problem of intracellular acyl-CoA accumulation. Treatments usually involve the restriction of dietary precursors and administration of agents like l-carnitine and glycine, which can accept the transfer of acyl groups from acyl-CoA, liberating CoASH. Many hereditary CASTOR patients are chronically ill, with persistent symptoms and continuously abnormal metabolites in blood and urine despite good compliance with treatment. Conversely, asymptomatic patients are also common in hereditary CASTOR conditions. Future challenges include the understanding of pathophysiologic mechanisms in CASTOR diseases, the discovery of reliable predictors of outcome in individual patients and the establishment of therapeutic trials with sufficient numbers of patients to permit solid therapeutic conclusions.

In vitro studies on the inhibition of colon cancer by butyrate and carnitine.

OBJECTIVE Epidemiologic studies support an association between diet and the incidence of colorectal cancer. Butyrate, a short-chain fatty acid present in dietary fiber and dairy products, is a potential anticarcinogenic compound. We previously showed that carnitine can enhance the bioavailability of butyrate in vivo. In the present study, we evaluated the effects of butyrate alone and in combination with carnitine on colon cancer cells in vitro, examining proliferation and apoptosis and the molecular mechanisms by which these nutrients may inhibit colon cancer. METHODS Caco-2 cells, a well-established cell model, were incubated with butyrate (2.5-20mM) with or without carnitine (10mM) for various incubation periods. Proliferation was measured by incorporation of (3)H-thymidine, and apoptosis was detected using flow cytometry, and then confirmed by analyzing the presence of single-strand DNA breaks typical of apoptotic cells. Prostaglandin E(2) production was assayed and Bcl-2 and cyclo-oxygenase-2 expressions were examined by western blotting. RESULTS Butyrate and carnitine inhibited Caco-2 cell proliferation (P<0.05) and induced apoptosis (P<0.05). Prostaglandin E(2) production was decreased in treated Caco-2 cells. At the molecular level, the expression of proapoptotic Bax and Bak proteins were increased in cells incubated with butyrate and carnitine, whereas expression of antiapoptotic Bcl-x(L) was decreased. Cyclo-oxygenase-2 expression was decreased in cells incubated with butyrate and carnitine. CONCLUSIONS Butyrate and carnitine inhibit human colon carcinoma cell proliferation and induce apoptosis in human colon carcinoma cells. This is accompanied by an appreciable alteration of the Bax-to-Bcl-x(L) and Bak-to-Bcl-x(L) ratios in favor of apoptosis. This study provides a scientific rationale to study the effects of carnitine and butyrate in colon cancer in vivo.

Regional amino acid neurotransmitter changes in brains of spf/Y mice with congenital ornithine transcarbamylase deficiency

Congenital deficiencies of the urea cycle enzyme ornithine transcarbamylase (OTC) result in chronic hyperammonemia and severe neurological dysfunction including seizures and mental retardation. As part of a series of studies to elucidate the pathophysiologic mechanisms responsible for the CNS consequences of OTC deficiency, concentrations of ammonia-related and neurotransmitter amino acids were measured as their o-phthalaldehyde derivatives using high performance liquid chromatography with fluorescence detection in regions of the brains of sparse-fur (spf) mice, a mutant with an X-linked inherited defect of OTC. Compared to CD-1/Y controls, the brains of spf/Y mutant mice contained significant alterations of several amino acids. A generalized, up to 2-fold, increase of brain glutamine was observed, consistent with the exposure of these brains to increased concentrations of ammonia. Significant increases of brain alanine were also observed and, together with previous reports of increased concentrations of α-ketoglutarate, are consistent with ammonia-induced inhibition of α-ketoglutarate dehydrogenase in the brains of spf/Y mice. Increased brain content of the excitatory amino acid aspartate coul be responsible for the seizures frequently encountered in congenital OTC deficiency.

Molecular basis of phenotypic variation in patients with argininemia

Argininemia is an autosomal recessive disorder caused by a deficiency in the liver-type arginase enzyme. Clinical manifestations include progressive spastic diplegia and mental retardation. While the quality of life can severely deteriorate in most such patients, some do show remarkable improvement in neurological symptoms while on controlled diets. We examined the thesis that differences in clinical responses to dietary treatment are based on molecular heterogeneity in mutant arginase alleles. Genomic DNAs from 11 patients with argininemia were examined using the polymerase chain reaction, cloning, and sequencing. Nine mutations representing 21/22 mutant alleles were identified in 11 patients with argininemia, and four of these mutations were expressed in vitro to determine the severity of enzymatic defects. We found that these mutations accounted for 64% of the mutant alleles in our patients. Based on findings in vitro expression tests, the mutations can be considered either severe or moderate. Patients with at least one moderate mutant allele responded well to dietary treatment; concentrations of plasma arginine were controlled within 300 μM. In contrast, patients with two severely mutated alleles did not respond to dietary treatment and plasma arginine was over 400 μM. Argininemia is heterogeneous at the molecular level. The degree of clinical improvement during dietary treatment is reflected in the concentration of arginine in plasma, as a measure of metabolic control. Plasma arginine levels during treatment is reflected in the concentration of arginine in plasma, as a measure of metabolic control. Plasma arginine levels during treatment correlated with types of molecular defects in the arginase genes.

Evidence for cholinergic neuronal loss in brain in congenital ornithine transcarbamylase deficiency

Congenital ornithine transcarbamylase (OTC) deficiency in humans is associated with seizures and mental retardation. As part of a series of studies to delineate the neurochemical features of OTC deficiency, activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), respectively, were measured in brain regions of the congenitally hyperammonemic sparse-fur (spf) mouse, a mutant with an X-linked inherited defect of OTC. ChAT activities were reduced by 63% (P < 0.01) in cerebral cortex of spf mice compared with CD-1/Y controls. Activities of the GABA nerve terminal marker enzyme, glutamic acid decarboxylase, on the other hand, were within normal limits. Using an immunohistochemical technique with a monoclonal antibody to ChAT, a significant loss of ChAT-positive neurons was observed throughout the cerebral cortex, septal area and diagonal band of spf mice. These results suggest that a loss of forebrain cholinergic neurons is a feature of congenital OTC deficiency in these mutants. Possible pathogenetic mechanisms responsible for the cholinergic neuronal loss in congenital OTC deficiency include neurotoxic effects of ammonia and accumulation of quinolinic acid.