In vitro effect of 4-nonylphenol on human chorionic gonadotropin (hCG) stimulated hormone secretion, cell viability and reactive oxygen species generation in mice Leydig cells.

Nonylphenol is considered an endocrine disruptor and has been reported to affect male reproductive functions. In our in vitro study, we evaluated the effects of 4-nonylphenol (4-NP) on cholesterol levels, hormone formation and viability in cultured Leydig cells from adult ICR male mice. We also determined the potential impact of 4-NP on generation of reactive oxygen species (ROS) after 44 h of cultivation. The cells were cultured with addition of 0.04; 0.2; 1.0; 2.5 and 5.0 µg/mL of 4-NP in the present of 1 IU/mL human chorionic gonadotropin (hCG) and compared to the control. The quantity of cholesterol was determined from culture medium using photometry. Determination of hormone production was performed by enzyme-linked immunosorbent assay. Metabolic activity assay was used for quantification of cell viability. The chemiluminescence technique, which uses a luminometer to measure reactive oxygen species, was employed. Applied doses of 4-NP (0.04-5.0 µg/mL) slight increase cholesterol levels and decrease production of dehydroepiandrosterone after 44 h of cultivation, but not significantly. Incubation of 4-NP treated cells with hCG significantly (P < 0.001) inhibited androstenedione, but not testosterone, formation at the highest concentration (5.0 µg/mL). The viability was significantly (P < 0.05); (P < 0.001) increased at 1.0; 2.5 and 5.0 µg/mL of 4-NP after 44 h treatment. Furthermore, 44 h treatment of 4-NP (0.04-5.0 µg/mL) caused significant (P < 0.001) intracellular accumulation of ROS in exposed cells. Taken together, the results of our in vitro study reported herein is consistent with the conclusion that 4-nonylphenol is able to influence hormonal profile, cell viability and generate ROS.

Effects of 4-nonylphenol on the steroidogenesis of human adrenocarcinoma cell line (NCI-H295R).

In this study, the human H295R adrenocarcinoma cell line was exposed to different concentrations (0.04, 0.2, 1.0, 2.5 or 5 µg/mL) of nonylphenol (NP) to investigate its impact on the inhibition or induction of the steroid hormones production during 48 h of in vitro culture. The hormone production was measured using ELISA kits. Results of this in vitro study suggest various effect of nonylphenol in relatively low concentrations on the selected steroid hormones production by the human H295R adrenocarcinoma cell line. The inhibiting impact on progesterone and androstenedione production was observed. The amount of progesterone was significantly decreased at 1.0, 2.5 and 5 µg/mL NP. Equally, the androstenedione production significantly decreased at 5 µg/mL NP. On the other hand, the amount of testosterone and 17ß-estradiol was induced after nonylphenol exposition. The significant increase of testosterone level was found out at treatment with 5 µg/mL NP. 17ß-estradiol production significantly increased at the doses of 2.5 and 5 µg/mL NP.

Cloning and characterization of a new polyketide synthase gene cluster in Streptomyces aureofaciens CCM 3239.

We cloned a new polyketide gene cluster, aur2, in Streptomyces aureofaciens CCM3239. Sequence analysis of the 9531-bp DNA fragment revealed 10 open reading frames, majority of which showed high similarity to the previously characterized type II polyketide synthase (PKS) genes. An unusual feature of the aur2 cluster is a disconnected organization of minimal PKS genes; ACP is located apart from the genes for ketosynthases KSalpha and KSbeta. The aur2 gene cluster was disrupted in S. aureofaciens CCM3239 by a homologous recombination, replacing the four genes (aur2A, E, F, G) including ketosynthase KSalpha, with antibiotic resistance marker gene. The disruption did not affect growth and differentiation, and disrupted strain produced spores with wild-type grey-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild type and aur2-disrupted strains did not reveal any difference in the pattern of antibacterial compounds.

Characterization of the polyketide spore pigment cluster whiESa in Streptomyces aureofaciens CCM3239.

A spore pigment polyketide gene cluster, whiESa, was cloned from Streptomyces aureofaciens CCM3239 using a probe from the S. coelicolor A3(2) whiE gene cluster. Sequence analysis of a 4,657-bp DNA fragment revealed five open reading frames with the highest similarity to the S. coelicolor A3(2) whiE locus responsible for spore pigment biosynthesis, with conservation of the size and position of the genes. The whiESa gene cluster was disrupted by a homologous recombination in S. aureofaciens CCM3239, replacing the most important whiESaIII gene encoding ketosynthase with a thiostrepton resistance gene. The mutation affected spore pigmentation. In contrast to wild-type grey-pink spore pigmentation, the mutant produced white spores, although overall spore morphology was not affected. Transcriptional analysis of whiESa revealed two divergently oriented promoters, whiESap1 and whiESap2, upstream of the whiESaI and whiESaVIII genes, respectively. Both promoters were developmentally regulated in S. aureofaciens CCM3239. They were induced at the late stages of differentiation, during sporulation of aerial hyphae and were dependent upon early sporulation-specific sigma factor sigma(RpoZ) and putative transcription factor WhiB. The level of the transcript originating from the whiESap2 promoter was substantially reduced in a sigF mutant of S. aureofaciens CCM3239, indicating its dependence upon the late sporulation sigma factor sigma(F). Comparison of the whiE promoters in three different spore pigment polyketide clusters revealed a highly conserved region upstream of the -35 promoter region that may bind a transcriptional regulator.

Cloning and characterization of a polyketide synthase gene cluster involved in biosynthesis of a proposed angucycline-like polyketide auricin in Streptomyces aureofaciens CCM 3239.

A new polyketide gene cluster, aur1, was identified in Streptomyces aureofaciens CCM3239 by using genes for the spore-pigment polyketide synthase of the Streptomyces coelicolor whiE operon as a probe. Sequence analysis of three overlapping DNA fragments (encompassing 15,100 bp) revealed 15 open reading frames, the majority of which showed high similarity to the previously characterized type II polyketide synthase genes. The highest similarity was to three Streptomyces polyketide gene clusters involved in biosynthesis of angucycline antibiotics, jadomycin, urdamycin and landomycin. The proposed S. aureofaciens ketosynthase (Aur1D) was phylogenetically more related to all known ketosynthases for polyketide antibiotics in Streptomyces than to spore-pigment ketosynthases. Interestingly, the aur1 gene cluster contained a gene encoding a proposed malonyl-CoA:ACP transacylase that has not been identified in any of the previously characterized type II polyketide synthase cluster. Transcriptional analysis of aur1 revealed a single promoter upstream the first open reading frame (the aur1A gene) that was active in all stages of differentiation with increased activity at the time of aerial mycelium formation. The aur1 gene cluster was disrupted by a homologous recombination, replacing the three genes (aur1B,C,D) including ketosynthase, with antibiotic resistance marker gene in S. aureofaciens chromosome. Disruption did not affect growth and differentiation; disrupted strain produced spores with wild-type gray-pink pigmentation. The biochromatographic analysis of the culture extracts from S. aureofaciens wild-type and aur1-disrupted strains revealed an antibacterial compound that was missing in the mutant. The results indicated a role of the S. aureofaciens aur1 gene cluster in biosynthesis of a polyketide secondary metabolite (which we named auricin), and not in the spore pigment biosynthesis.