Hepatotoxicity of bisphenol A under conditions of differential supplementation with retinoids.

Classical xenoestrogenic in vivo effects of bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) are well-described in the literature, however the molecular mechanisms of BPA-induced hepatotoxicity are not fully characterized. The work is aimed to assess biochemical markers of BPA induced hepatotoxicity under conditions of differential supplementation with retinoids. We demonstrate that the absence of hepatic retinyl esters as the main form of vitamin A storage provides for a resistance to BPA induced liver damage. Retinoid supplementation increases the hepatotoxic effects of bisphenol A, evidenced in higher indexes of oxidative damage of lipids, proteins and non-protein thiol groups as well as increase of serum alanine aminotransferase activity and myeloperoxidase activity in liver parenchyma. The absence of hepatotoxicity signs when hepatic retinoid stores are depleted and their presence during normal or excessive retinoid supplementation suggest that hepatic retinoid availability is one of the factors determining the hepatotoxicity of bisphenol A.

Molecular genetics of naringenin biosynthesis, a typical plant secondary metabolite produced by Streptomyces clavuligerus.

BACKGROUND: Some types of flavonoid intermediates seemed to be restricted to plants. Naringenin is a typical plant metabolite, that has never been reported to be produced in prokariotes. Naringenin is formed by the action of a chalcone synthase using as starter 4-coumaroyl-CoA, which in dicotyledonous plants derives from phenylalanine by the action of a phenylalanine ammonia lyase. RESULTS: A compound produced by Streptomyces clavuligerus has been identified by LC-MS and NMR as naringenin and coelutes in HPLC with a naringenin standard. Genome mining of S. clavuligerus revealed the presence of a gene for a chalcone synthase (ncs), side by side to a gene encoding a P450 cytochrome (ncyP) and separated from a gene encoding a Pal/Tal ammonia lyase (tal). Deletion of any of these genes results in naringenin non producer mutants. Complementation with the deleted gene restores naringenin production in the transformants. Furthermore, naringenin production increases in cultures supplemented with phenylalanine or tyrosine. CONCLUSION: This is the first time that naringenin is reported to be produced naturally in a prokariote. Interestingly three non-clustered genes are involved in naringenin production, which is unusual for secondary metabolites. A tentative pathway for naringenin biosynthesis has been proposed.

Effect of Deletion of Ghrelin-O-Acyltransferase on the Pulsatile Release of Growth Hormone in Mice.

Ghrelin, a gut hormone originating from the post-translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin-O-acyltransferase (GOAT), conferring selective binding to the GHS-R1a receptor via acylated ghrelin. Complete loss of preproghrelin-derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl-ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat(-/-) mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat(-/-) mice compared to age-matched wild-type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH-dependent liver genes. Of interest, circulating levels of insulin-like growth factor (IGF)-1 were elevated in goat(-/-) mice. This rise in circulating levels of IGF-1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF-1 release in goat(-/-) mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF-1 release and growth in goat(-/-) mice.

Acylated ghrelin is not required for the surge in pituitary growth hormone observed in pregnant mice.

OBJECTIVE: Ghrelin is produced by the stomach, hypothalamus and pituitary. It circulates as acylated ghrelin (AG, which stimulates growth hormone (GH) secretion) and unacylated ghrelin (UAG). Acylation is mediated by the enzyme ghrelin O-acyltransferase (GOAT). In mice, pregnancy is associated with a marked increase in circulating pituitary GH. We investigated the role of AG and UAG in the surge of plasma GH concentrations in pregnant mice at the end of pregnancy. DESIGN: Using a mouse model generated on a C57BL/6 background (wild type, WT) in which the GOAT gene has been deleted (KO), we measured plasma AG, UAG and GH concentrations and tissue (stomach, pituitary and hypothalamus) preproghrelin and GOAT mRNA in non-pregnant (NP) and pregnant (P), WT and KO mice. RESULTS: GOAT deletion was associated with undetectable concentrations of AG. UAG concentrations were similar in all groups. In both WT and KO animals, mean GH concentrations increased 30 to 50 times during pregnancy. There was a tendency toward lower median GH concentrations in KO (301 ng/mL) compared to WT (428 ng/mL) mice (p=0.059). Preproghrelin expression was not affected by GOAT deletion or by pregnancy in the stomach. In contrast, pituitary and hypothalamic ghrelin gene expression were lower in KO-NP and KO-P mice compared to their WT counterparts. CONCLUSION: The complete absence of ghrelin acylation, which is associated with undetectable AG concentrations, does not prevent the marked increase in pituitary GH concentrations observed in pregnant mice, suggesting that AG is not the major mediator of GH secretion during pregnancy.

Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis.

Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: first the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the ATS2 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the ATS2 cDNA into E. coli JC 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild type growth at high temperature. A green-fluorescent protein fusion with ATS2 localized to the chloroplast. Disruption of the ATS2 gene of Arabidopsis by T-DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in ATS2 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form.

Deficiency of acyl coenzyme a:diacylglycerol acyltransferase 1 increases leptin sensitivity in murine obesity models.

Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known enzymes that catalyze the final step in mammalian triglyceride synthesis. We have reported that DGAT1-deficient mice have increased insulin and leptin sensitivity, likely accounting for their protection against diet-induced obesity and insulin resistance. Here we show that DGAT1 deficiency enhanced the response to peripheral leptin infusion in Agouti yellow and leptin-deficient (ob/ob) mice, two genetic models of obesity and insulin resistance. Interestingly, DGAT1 deficiency did not enhance the response to intracerebroventricular leptin infusion. Moreover, DGAT1 deficiency did not alter the expression of key hypothalamic genes involved in leptin signaling or in the regulation of food intake and energy expenditure. Thus, the leptin-sensitizing effect of DGAT1 deficiency is present in both leptin-resistant and leptin-deficient genetic models of obesity and may occur in part by enhancing the effects of leptin in peripheral tissues.

Emotional distress induced rhabdomyolysis in an individual with carnitine palmitoly-transferase deficiency.

A 48-year-old male patient with underlying CPT II enzyme deficiency is described. Emotional stress appeared to precipitate recurrent myalgias, rhabdomyolysis and reversible renal impairment over a 40-year period. Our search of the English literature indicates this to be the first time that the emotional stress has been documented to precipitate the CPT II syndrome. Although the pathogenesis of this syndrome has yet to be established, existing knowledge is briefly reviewed and the likely metabolic and neuroendocrine mechanisms which link emotional stress to muscle metabolism are examined. These mechanisms influence the extent of lipolysis or glycolysis that occurs during the process of muscle ATP generation. It is suggested that neuroendocrine and other stress related changes which favour lipolysis over glycolysis adversely affect muscle energy metabolism in patients whose mitochondria are deficient in CPT II enzyme. Possible treatment strategies are those that favour glycolysis over fatty acid metabolism and include a variety of ways of modulating sympathetic and parasympathetic tone. The use of carbohydrate supplementation beta-blockers and anxiolytic agents is discussed.

Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.

We have developed a two-step selection protocol to generate a population of Chinese hamster ovary (CHO) cell variants that are plasmalogen-deficient, but contain intact, functional peroxisomes (plasmalogen-/peroxisome+). This involved sequential exposures of a mutagenized cell population to photodynamic damage by using two different pyrene-labelled sensors, 9-(1'-pyrene)nonanol and 12-(1'-pyrene)dodecanoic acid. By this procedure we generated several isolates, all except one of which displayed a severe decrease in plasmalogen biosynthesis. Further characterization of one of the plasmalogen-deficient isolates, NRel-4, showed that it contained intact, functional peroxisomes. Whole-cell homogenates from NRel-4 displayed severely decreased dihydroxyacetone phosphate acyltransferase, which catalyses the first step in plasmalogen biosynthesis. NRel-4 and another, recently described, plasmalogen-deficient cell line, NZel-1 [Nagan, Hajra, Das, Moser, Moser, Lazarow, Purdue and Zoeller (1997) Proc. Natl. Acad. Sci. U.S. A. 94, 4475-4480] were hypersensitive to singlet oxygen, supporting the notion of plasmalogens as radical oxygen scavengers. Wild-type-like resistance could be conferred on NRel-4 upon restoration of plasmalogen content by supplementation with a bypass compound, sn-1-hexadecylglycerol. NRel-4 and other plasmalogen-/peroxisome+ strains will allow us to examine further the role of ether lipids in cellular functions without complications associated with peroxisome deficiency, and might serve as an animal cell model for certain forms of the human genetic disorder rhizomelic chondrodysplasia punctata.

Mutagenic analysis of 2,3-diaminophenazine and 2-amino-3-hydroxyphenazine in Salmonella strains expressing different levels of O-acetyltransferase with and without plant and mammalian activation.

2,3-Diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) are products generated from oxidative-type phenylenediamine hair dyes and are also present in pesticide formulations as contaminants. Earlier studies demonstrated that DAP and AHP were mutagenic in Salmonella typhimurium strains after mammalian microsomal activation. Plant systems can activate structurally similar arylamines. S. typhimurium strains have been developed that express elevated levels of acetyl-CoA: N-hydroxyarylamine O-acetyltransferase (OAT). O-acetyltransferase expression is necessary for the generation of the ultimate arylamine promutagen after plant activation. A number of arylamines including 2-aminofluorene, benzidine and 4-aminobiphenyl were activated by plant cells into mutagens in the OAT over-expressing S. typhimurium strain, YG1024. The objectives of this research were to examine the mutagenicity of DAP and AHP with mammalian or plant activation in Salmonella strains with different acetyltransferase activities. The hypothesis tested was whether and to what degree a metabolite of DAP or AHP could serve as a substrate for bacterial O-acetyltransferase and induce mutation in Salmonella. DAP and AHP without activation induced both frameshift and base pair substitution mutations in S. typhimurium strains that exhibited elevated levels of O-acetyltransferase activity. The mutagenicity of DAP and AHP were greatly enhanced with mammalian hepatic microsomal activation resulting in a preferential induction of frameshift mutations. With the hisD3052 allele as the gene target, S9-activated DAP induced frameshift mutations in YG1024 and TA98 as well as the OAT deficient strain TA98/1,8-DNP6. S9-activated AHP induced mutation only in the OAT over-expressing strain, YG1024. With the hisG46 allele, O-acetyltransferase activity was necessary for the metabolism of DAP and AHP to products that induce base pair substitution mutations. An intriguing finding of this work was the antimutagenic capacity of TX1MX, a plant cell-free activation mixture. TX1MX repressed the mutagenic activity of DAP and AHP at frameshift and base pair substitution mutation targets.