Modulation of N-methyl-N-nitrosourea-induced crypt restricted metallothionein immunopositivity in mouse colon by a non-genotoxic diet-related chemical.

Red meat consumption is associated with endogenous metabolic generation of mutagenic N-nitroso compounds (NOC) and may be implicated in causation of colorectal cancer. Assessment of a biologically relevant dose of NOCs is hampered by imperfect understanding of NOC interactions with other dietary components. This study tests the hypothesis that NOC effects upon mutational biomarkers in mouse colon may be modulated by a non-genotoxic diet-related compound. N-methyl-N-nitrosourea (MNU) and undegraded lambda carrageenan (lambdaCgN) were selected as test chemicals, representing a NOC and a non-genotoxic agent, respectively. Study end-points included (i) DNA adduct formation and (ii) metallothionein (MT) crypt restricted immunopositivity indices (MTCRII) which are considered representative of crypt stem cell mutations. Frequency and size of MT immunopositive foci as well as total number of MT immunopositive crypts were assessed. Biologically effective doses of MNU and lambdaCgN were determined in model validation studies and the agents were then tested alone and in combination. Continuous lambdaCgN treatment for 10 weeks induced significantly greater colonic mucosal injury than a drinking water control. In combined treatment regimens, lambdaCgN treatment did not significantly affect MNU-induced DNA adduct formation. However, combinations of lambdaCgN with MNU significantly increased MTCRII in excess of those induced by MNU alone. Recurrent or continuous lambdaCgN regimens had greater interactive effects with MNU upon MTCRII than short-term lambdaCgN treatment. This study has shown that exposure to a non-genotoxic diet-related compound (lambdaCgN) modulates the effective NOC dosimetry for induction of MT crypt restricted immunopositivity.

Effects of cryopreservation on testicular sperm nuclear DNA fragmentation and its relationship with assisted conception outcome following ICSI with testicular spermatozoa.

The objective of the study was to investigate the effects of freeze-thawing on testicular sperm DNA fragmentation, fertilization rates and pregnancy rates following intracytoplasmic sperm injection with testicular spermatozoa (TESE). This ongoing prospective study included 88 couples attending for infertility treatment where the man presented with obstructive azoospermia at the Regional Fertility Centre, Belfast, UK. Patients were allocated to receive TESE treatment with fresh or freeze-thawed spermatozoa. Sperm aliquots were stored in liquid nitrogen at -196 degrees C following static phase vapour cooling or cooling at controlled rates using a programmable freezer. Samples were thawed at either room temperature or 37 degrees C. Sperm nuclear DNA; assessed by the alkaline Comet assay, was significantly damaged by slow freezing followed by fast thawing. Pregnancies were more likely to be achieved with spermatozoa displaying markedly less DNA damage. However, no differences were observed in the fertilization rates, the number of blastomeres or the cumulative embryo score between TESE cycles using either fresh or frozen thawed testicular spermatozoa. The pregnancy rates tended to be higher following fresh TESE cycles (30%) compared with TESE cycles using frozen-thawed testicular spermatozoa (26%), although this difference did not reach statistical significance. It is concluded that cryopreservation of testicular spermatozoa may reduce pregnancy rates, although this will only be confirmed by a much larger multi-centre trial.