High expression of the mRNA of cytochrome P450 and phase II enzymes in the lung and kidney tissues of cattle

The objective of this study was to investigate the tissue-specific mRNA expression of different cytochrome P450 (CYP) isoforms, UDP glucuronsyl transferase 1A1 (UGT1A1) and glutathione-S-transferase (GSTA1) in the different tissues (liver, mammary gland, lungs, spleen, kidney cortex, heart, masseter muscle and tongue) of cattle, using quantitative real-time polymerase chain reaction (qPCR). CYP1A1-like mRNA was expressed in all of the tissues examined, including the liver, with the highest expression level in the kidney. CYP1A2-, 2E1- and 3A4-like mRNAs were only expressed hepatically. Interestingly, significant expression of CYP2B6-like mRNA was recorded in the lung tissue, while CYP2C9-like mRNA was expressed in the liver and kidney tissues of the cattle examined. UGT1A1- and GSTA1-like mRNAs were expressed in all of the examined tissues, except the mammary glands, and the highest expression levels were recorded in the kidney. The high expression of UGT1A1 in the lung tissue and GSTA1 in the liver tissue was unique to cattle; this has not been reported for rats or mice. The findings of this study strongly suggest that the liver, kidneys and lungs of cattle are the major organs contributing to xenobiotics metabolism.     

Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.

Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.     

Tissue-specific, inducible, and hormonal control of the human UDP-glucuronosyltransferase-1 (UGT1) locus

The human UDP-glucuronosyltransferase 1 (UGT1) locus spans nearly 200 kb on chromosome 2 and encodes nine UGT1A proteins that play a prominent role in drug and xenobiotic metabolism. Transgenic UGT1 (Tg-UGT1) mice have been created, and it has been demonstrated that tissue-specific and xenobiotic receptor control of the UGT1A genes is influenced through circulating humoral factors. In Tg-UGT1 mice, the UGT1A proteins are differentially expressed in the liver and gastrointestinal tract. Gene expression profiles confirmed that all of the UGT1A genes can be targeted for regulation by the pregnane X receptor activator pregnenolone-16alpha-carbonitrile (PCN) or the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, the selective induction of glucuronidation activity toward lamotrigine, ethinyl estradiol, chenodeoxycholic acid, and lithocholic acid by either PCN or TCDD in small intestine from Tg-UGT1 mice corresponded to expression of the locus in this tissue. Induction of UGT1A1 by PCN and TCDD is believed to be highly dependent upon glucocorticoids, because submicromolar concentrations of dexamethasone actively promote PCN and TCDD induction of UGT1A1 in Tg-UGT1 primary hepatocytes. The role of hormonal control of the UGT1 locus was further verified in pregnant and nursing Tg-UGT1 mice. In maternal 14-day post-conception Tg-UGT1mice, liver UGT1A1, UGT1A4, and UGT1A6 were induced, with the levels returning to near normal by birth. However, maternal liver UGT1A4 and UGT1A6 were dramatically elevated and maintained after birth, indicating that these proteins may play a critical role in maternal metabolism during lactation. With expression of the UGT1 locus confirmed in a variety of mouse tissues, these results suggested that the Tg-UGT1 mice will be a useful model to examine the regulatory and functional properties of human glucuronidation.     

A single mouse alpha-amylase gene specifies two different tissue-specific mRNAs

The alpha-amylase mRNAs which accumulate in two different tissues of the mouse, the salivary gland and the liver, are identical except for their 5' non-translated sequences: the 5' terminal 158 nucleotides of the major liver alpha-amylase mRNA are unrelated to the 5' terminal 47 nucleotides found in its salivary gland counterpart. DNA that specifies the 5'terminal one-quarter of these mRNAs has been isolated through genomic cloning and sequenced. The initial 161 nucleotides of the liver alpha-amylase mRNA are specified by DNA sequences that lie 4.5 kb upstream from those for the common body of the two mRNAs. In contrast, the 5' terminal 50 nucleotides of the salivary gland alpha-amylase mRNA are found 7.5 kb from sequences that the two mRNAs share in the genome. These cloned DNA sequences occur once per haploid genome, indicating that both the salivary gland and liver alpha-amylase mRNAs are transcribed from the same gene (Amy1A). Since no rearrangement of these DNA sequences can be detected among mouse sperm, salivary gland or liver preparations, gross rearrangement does not account for the tissue-specific pattern of expression observed for Amy1A. Rather, these data indicate that the salivary gland and liver alpha-amylase mRNAs are differentially transcribed and/or processed from identical DNA sequences in different tissues.     

Glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38) by the human UDP-glucuronosyltransferases encoded at the UGT1 locus.

7-Ethyl-10-hydroxycamptothecin (SN-38) is a very promising anticancer drug used for the treatment of metastatic colonrectal cancer. SN-38 is the active metabolite of irinotecan, a semisynthetic anticancer drug derived from 20(S)camptothecin. In this study, we examined the potential for each of the UGT1-encoded isoforms (UGT1A1 and UGT1A3 through UGT1A10) to glucuronidate SN-38. The amount of specific protein for each isoform was determined by Western blot analysis. Although UGT1A1 was previously shown to metabolize this drug, the results of this study show that UGT1A7 glucuronidates this chemical at a 9- to 21-fold higher level at pH 6. 4 and pH 7.6, respectively, than that by UGT1A1. The activity of UGT1A7 is from 8.4- to 19-fold higher at pH 6.4 and 12- to 40-fold higher at pH 7.6 than that by the other 7 UGT1 encoded isoforms. UGT1A7 glucuronidates SN-38 with an apparent Km of 5 microM. Hence, the distribution of this isoform in the gastrointestinal tract has the potential to impact the effectiveness of this chemotherapeutic agent.