Measurement of intracellular LDH activity in 96-well cultures: A rapid and automated assay for cytotoxicity studies

Summary The present paper describes a rapid, easy, sensitive, and automated spectrophotometric assay for intracellular lactate dehydrogenase (LDH) measurement in 96-well plates of adherent cells for cytotoxicity studies. The procedure involves “in situ” homogenization of cells, followed by measurement of LDH activity with a colorimetric method based on the reduction of a tetrazolium salt to a violet formazan by the NADH formed by LDH. Color intensity can be measured in conventional ELISA readers, and the data can be fed to an “on line” computer for rapid processing. The color absorbance measured is time- and enzyme-concentration dependent. LDH activity measured with this micromethod is coincident with that measured in larger culture plates after individual homogenization and conventional LDH measurement. The advantages of this method are the smaller number of cells required, easy automation, drastic reduction of time for processing individual wells, and the possibility of examining multiple variables in the same experiment.     

Activity of cytochrome P450 1A in the liver of CBA and CC57BR mice after prolonged repeated induction witho-aminoazotoluene

Cytochrome P450 1A activity in the liver of CBA and CC57BR mice sensitive and resistant to hepatocarcinogenic effects ofo-aminoazotoluene, respectively, was measured after prolonged (for several months) repeated administration of this agent. Repeated and single injections of this carcinogen produced similar effects on microsomal monooxygenases in CC57BR mice, but caused different changes in cytochrome P450 1A activity in the liver of CBA mice. Hence, enhanced inducibility of cytochrome P450 1A in CBA mice after prolonged treatment witho-aminoazotoluence probably contributes to their sensitivity to hepatocarcinogenic effects of this agent. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny Vol. 129, No. 3, pp. 280–282, March, 2000     

The CCHCR1 (HCR) gene is relevant for skin steroidogenesis and downregulated in cultured psoriatic keratinocytes

The HCR gene, officially called Coiled-Coil alpha-Helical Rod protein 1 (CCHCR1), located within the major psoriasis susceptibility locus PSORS1, is a plausible candidate gene for the risk effect. Recently, CCHCR1 was shown to promote steroidogenesis by interacting with the steroidogenic acute regulator protein (StAR). Here, we examined the role of CCHCR1 in psoriasis and cutaneous steroid metabolism. We found that CCHCR1 and StAR are expressed in basal keratinocytes in overlapping areas of the human skin, and CCHCR1 stimulated pregnenolone production in steroidogenesis assay. Overexpression of either the CCHCR1*WWCC risk allele or the non-risk allele enhanced steroid synthesis in vitro. Furthermore, the cytochrome P450scc enzyme was expressed in human keratinocytes and was induced by forskolin, a known activator of steroidogenesis, and forskolin also upregulated CCHCR1. CCHCR1 has an altered expression pattern in lesional psoriatic skin compared to normal healthy skin, suggesting its dysregulation in psoriasis. We found that the expression of CCHCR1 is downregulated twofold at the mRNA level in cultured non-lesional psoriatic keratinocytes when compared to non-psoriatic healthy cells. Our results also suggest a connection between CCHCR1 and vitamin D metabolism in keratinocytes. The expression of the vitamin D receptor (VDR) gene was lower in non-lesional psoriatic keratinocytes than in healthy cells. Furthermore, Vdr expression was downregulated in the keratinocytes of mice overexpressing the CCHCR1*WWCC risk allele when compared to keratinocytes from mice with the non-risk allele of CCHCR1. Finally, we demonstrate that other agents relevant for psoriasis and/or the regulation of steroidogenesis influence CCHCR1 expression in keratinocytes, including insulin, EGF, cholesterol, estrogen, and cyclosporin A. Taken the role of steroid hormones, including vitamin D and estrogen, in cell proliferation, epidermal barrier homeostasis, differentiation, and immune response, our results suggest a role for CCHCR1 in the pathogenesis of psoriasis via the regulation of skin steroid metabolism.     

Activity and inducibility of cytochrome P-450-1A in the liver of mice with different sensitivity to hepatocarcinogenic effect of o-aminoazotoluene

Specific activity of cytochrome P-450-1a is detected in the liver of 7 inbred mouse strains sensitive (SWR, C3HA, DD, and CBA) and resistant (AKR, CC57BR, and C57BL) to o-aminoazotoluene-induced hepatocarcinogenesis: 7-ethoxyresorufin-O-diethylase and 7-methoxyresorufin-O-dimethylase, induced by benzo(a)pyrene and o-aminoazotoluene. Mice sensitive (CC57BR and C57BL) and resistant (AKR) to P-450-1a induction were resistant to induction of liver tumor and, similarly, mice both resistant (SWR and DD) and sensitive (C3HA and CBA) to P-450-1a induction were sensitive to hepatic tumor induction. Therefore, there was no correlation between inducibility of cytochrome P-450-1a and sensitivity to o-aminoazotoluene-induced hepatocarcinogenesis at the initial stages of chemical carcinogenesis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 8, pp. 201–203, August, 1998