Regulation of cutaneous drug-metabolizing enzymes and cytoprotective gene expression by topical drugs in human skin in vivo.

BACKGROUND: Individuality in the expression and regulation of hepatic drug-metabolizing enzymes (DMEs) and cytoprotective (CP) genes is an important determinant of treatment response. There is increasing evidence that many DMEs and CP genes are also expressed in human skin. Responses to topical drugs used to treat common skin diseases, such as psoriasis, are unpredictable and may potentially be rationalized, at least in part, by interindividual differences in cutaneous DME and CP gene expression. OBJECTIVES: We investigated whether three topical drugs [coal tar, all-trans retinoic acid (atRA) and clobetasol 17-propionate] used in routine clinical practice modulated the expression of a variety of DME and CP genes [cytochrome P450s, glutathione S-transferases (GSTs) and drug transporters] in healthy human skin in vivo. METHODS: Healthy adult volunteers (n = 30) were invited to participate in the study. Each subject was randomly allocated to receive two of the three study chemicals and one control site application. Crude coal tar (n = 13), atRA (n = 14) or clobetasol 17-propionate (n = 10) was applied under occlusion to photoprotected buttock skin for 96 h. A vehicle control (white soft paraffin) was also applied under the same conditions at an adjacent site in all subjects. Full-thickness punch biopsies (4-mm diameter) were then taken from treated and control sites. Total RNA was extracted and reverse transcribed into cDNA, which was used as a template in subsequent real-time polymerase chain reaction analysis, where fluorescent output was directly proportional to input cDNA concentration. Triplicate measurements of skin mRNA expression were made from each sample, and the arithmetic mean values taken. After logarithmic transformation, the paired t-test was used to compare values between treated and control skin. RESULTS: Cytochrome P450s CYP1A1, CYP1A2, CYP1B1, CYP2C18, quinone reductase (NQO-1), GSTP1, gamma-glutamyl cysteine synthetase (gamma-GCS), glutathione peroxidase-1 (GPx-1), cyclooxygenase-2 (COX-2) and haem oxygenase-1 (HO-1) were induced by coal tar; CYP26, NADPH P450 reductase (CPR), GSTP1 and HO-1 by atRA; and CYP3A5 by clobetasol 17-propionate. In contrast, CYP1A1 and CYP1A2 expression was suppressed by atRA, and gamma-GCS and MRP1 by clobetasol 17-propionate. Marked interindividual variation in gene regulation by topical drugs was seen for the majority of genes examined. CONCLUSIONS: These data demonstrate that topical drugs can modulate DME gene expression in human skin in vivo and indicate that variation in the expression and regulation of these genes may be a determinant of individuality in response to topical therapies for common skin diseases.

Cloning and expression of two isoforms of guanylate cyclase C (GC-C) from the European eel (Anguilla anguilla).

Complementary DNA fragments for two isoforms of particulate guanylate cyclase C (GC-C) were cloned from the intestine of the European eel (Anguilla anguilla). Both isoforms exhibited higher nucleotide and amino acid sequence homologies to members of the GC-C family from other species than the related guanylate cyclase A or B (GC-A or GC-B) isoforms from the eel. Northern blots indicated that probes for both isoforms, termed GC-C1 and GC-C2, selectively hybridised to 4.8-kb transcripts in the intestine and the kidney. Expression of the GC-C2 transcript in the intestine was increased by 100% following the transfer of yellow FW-acclimated eels to SW. Likewise developmental maturation of yellow eels into pre-migratory silver eels resulted in a significant increase (60%) in the intestinal expression of GC-C2. No changes in expression of GC-C2 were seen in the kidney under any condition. RT-PCR indicated that the GC-C2 isoform is only expressed in anterior and mid-gut segments in FW-acclimated yellow eels. However, expression is also extended to the posterior gut segment when yellow eels are acclimated to SW or following developmental transformation into silver eels.

Cloning and Expression of Guanylin from the European eel (Anguilla anguilla).

Extracts of intestinal epithelia from the European eel (Anguilla anguilla) stimulated cGMP production in the T84 human colon carcinoma cell line which suggested the presence of a guanylin-like peptide in this teleost fish. Degenerate oligonucleotide primers were subsequently used in RT-PCR resulting in the amplification, cloning, and sequencing of two cDNAs which represent possible 5' spliceoforms of an eel homologue of the mammalian peptide, guanylin. Northern blotting indicated that the main site of expression of the eel peptide is in the intestine with much lower signals also detected in the kidney. Intestinal expression of guanylin mRNA is up-regulated in both nonmigratory "yellow" and the more sexually mature, migratory "silver" eels following acclimation to the seawater environment. These results suggest that this peptide signalling system may play a role in osmoregulation in euryhaline teleost fish during migration between the marine and freshwater environments.