title: Lymphocyte Hprt mutant frequency and sperm toxicity in C57BL/6 mice treated chronically with Azathioprine.
abstract: Many patients undergoing chronic therapy with the purine analogue Azathioprine (Aza) have highly elevated HPRT lymphocyte mutant frequencies (MFs), and it is likely that these increases are due to selection of pre-existing HPRT mutant lymphocytes. A similar selection in germ cells might result in an increased frequency of the Lesch-Nyhan syndrome. In this study, a mouse model for Aza mutant selection was developed and Aza toxicity was evaluated in the germ cells of treated mice. Groups of 20 male C57BL/6 mice were treated by gavage three times/week with 0, 5, 10, 25, 50, or 100mg/kg Aza, and three to eight mice from each group were sacrificed at various times for up to 23 weeks. Mice treated with 25-100mg/kg Aza were all dead by 14 weeks of treatment. Hprt lymphocyte MF assays indicated that the treated mice had reduced numbers of spleen lymphocytes. Most treated mice had Hprt MFs similar to those of control mice (2.1+/-1.6 x 10(-6)), however, highly elevated MFs were detected in one out of three mice given 5mg/kg for 10 weeks, one out of three mice given 10mg/kg for 10 weeks, and one out of eight mice given 10mg/kg for 23 weeks (e.g., 233 x 10(-6) after 10 weeks of 5mg/kg). Sequence analysis of Hprt cDNA indicated that all mutant clones from one of these mice had a T-->A transversion in the initiation codon. Multiplex-PCR on mutant clones from the other two mice indicated that all the clones from one had a deletion of Hprt exons 2 and 3, while most of the mutants from the other had lost all of the Hprt exons. Measurements of testicular weight, and of sperm count, viability, morphology, and motility found that Aza produced low levels of toxicity in sperm, with the most consistent effect being a reduction in the testicular weight. The data suggest that mice chronically treated with 5 and 10mg/kg Aza (doses similar to those used in humans) have elevated Hprt MFs due to clonal amplification of selected Hprt mutants. The results also suggest that mice treated with these doses of Aza retain reasonable fertility, and will be useful for breeding experiments to examine the possibility of increasing the germ-line transmission of Hprt mutations.
authors.affiliation: University of Arkansas for Medical Sciences, Department of Pharmacology and Toxicology, Little Rock, AR, USA.
authors.forename: Sachin V
authors.initials: SV
authors.lastname: Bendre
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Shaddock
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Patton
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Dobrovolsky
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Albertini
authors.forename: Robert H
authors.initials: RH
authors.lastname: Heflich
mesh_terms: D000818:Animals; D001379:Azathioprine / Q000737:chemistry / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005091:Exons; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008214:Lymphocytes / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015394:Molecular Structure; D009153:Mutagens / Q000737:chemistry / Q000633:toxicity*; D009154:Mutation; D009929:Organ Size / Q000187:drug effects; D017384:Sequence Deletion; D013094:Spermatozoa / Q000187:drug effects*; D013154:Spleen / Q000166:cytology; D036401:Toxicity Tests, Chronic
chemical_list: D009153:Mutagens; D007041:Hypoxanthine Phosphoribosyltransferase; D001379:Azathioprine