The effect of benzo(a)pyrene on porcine urinary bladder epithelial cells analyzed for the expression of selected genes and cellular toxicological endpoints.

Consumption of tobacco products is the most relevant risk factor for the development of bladder cancer beside occupational contributions. In order to investigate mechanisms of tobacco smoke components in bladder carcinogenesis we have introduced a primary epithelial cell culture system derived from porcine urinary bladder as a suitable representative for the corresponding human tissue under physiological conditions. Two independent readouts were selected as markers for genotoxic events. Changes in the expression level of several toxicologically relevant genes should serve as indicators for early response, while classical genotoxic endpoints monitored manifested damages. Here, we present the first results of our study with benzo(a)pyrene (BaP) as a member of polycyclic aromatic hydrocarbons (PAHs) found in tobacco smoke. Cells treated with BaP show a dramatic increase in the expression of CYP1A1 that appears to be both indicator of and contributor for BaP toxicity. Genes coding for other proteins relevant in xenobiotic metabolism, signal transduction or tumor suppression show moderate effects or no enhancement of their expression levels. Comet assay and micronucleus test did show a significant, dose-dependent increase in DNA damages or aberrations after cell division. While these effects are conforming to the response at the mRNA expression level, they are less pronounced and require rather higher dosages of the chemical.

A role for nitrilase 3 in the regulation of root morphology in sulphur-starving Arabidopsis thaliana.

Arabidopsis thaliana expresses four nitrilases, three of which (NIT1, NIT2 and NIT3) are able to convert indole-3-acetonitrile to indole-3-acetic acid (IAA), the plant growth hormone, while the isozyme NIT4 is a beta-cyano-l-alanine hydratase/nitrilase. NIT3 promoter activity is marginal in leaves or roots of vegetative plants and undetectable in bolting and flowering plants, but its level increases strongly when plants experience sulphur deprivation. No other nitrilase genes respond to sulphur supply/deficiency. Neither N- nor P-deprivation cause detectable changes in NIT3 promoter activity. In transgenic plants expressing uidA under the control of the NIT3 promoter (NIT3p::uidA), sulphate deprivation leads to the appearance of beta-glucuronidase activity in shoots and particularly in roots, most strongly in the conductive tissues and lateral root primordia. Deletion analysis allowed localization of the sulphur-responsive element to a 317 bp segment of the NIT3 promoter encompassing nt -2151 to -1834 upstream of the transcriptional start point. Both nitrilase polypeptide and nitrilase activity were also induced by sulphur starvation. NIT3 promoter activity was strongly induced by O-acetylserine, suggesting that, as is the case with enzymes of sulphate assimilation, sulphate deficiency may be communicated to NIT3 via an increase in the level of the cysteine precursor, O-acetylserine. During sulphur deprivation, a preferential depletion of the pool of the indole-3-acetonitrile precursor glucobrassicin compared with that of total glucosinolates was noticed. In the absence of an external sulphate supply, plants developed longer roots with a higher number of lateral roots. The increased growth of the root system occurred at the expense of shoot growth which was retarded under conditions of sulphur starvation. Taken together, these results suggest that a regulatory loop appears to exist by which sulphate deficiency, through an increase in glucobrassicin turnover and nitrilase 3 accumulation, initiates the production of extra auxin leading to increased root growth and branching, thus allowing the root system to penetrate new areas of soil effectively to gain access to fresh supplies of sulphur.