In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay

Abstract

Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P < 0.05). B[a]P-related DNA adducts were detected at increased levels in smokers as determined by immunostaining. Direct exposure of mature sperm cells to B[a]P (10 or 25 μM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 μM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE–DNA adducts as measured with 32P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies.

Single Layer Centrifugation with Androcoll-E&lt;sup&gt;TM&lt;/sup&gt; improved progressive motility and percentage of live spermatozoa with intact acrosome of chilled stallion semen but did not have an effect on DNA integrity

“…The protocol was based on Sipinen et al [19]. Slides were precoated with agarose by dipping into a vertical staining jar of melted 1% standard agarose in water.…”

Combined Effects of Dibutyl Phthalate (DBP) and Benzo(a)Pyrene on Fertility in Male Rats

“…Benzo(a)pyrene (Bap), a prototype of polycyclic aromatic hydrocarbon (PAH) that is released into the environment by industrial emission, has attracted worldwide attention as its toxic effect on male reproduction. It has been demonstrated that Bap can cause seminiferous tubules malformation, change the epididymis function and enzyme activity in testicular cells, induce DNA damage and synthesis inhibition in spermatogenic cells, and decrease serum testosterone level and the amount of spermatogenic cells and sperms (Zhu et al, 2010;Sipinen et al, 2010;Inyang et al, 2003;Mohammad Shah Alam et al, 2010;Ramesh et al, 2008).…”

Tobacco consumption and benzo(a)pyrene-diol-epoxide–DNA adducts in spermatozoa: in smokers, swim-up procedure selects spermatozoa with decreased DNA damage

“…In studies that quantify BPDE-DNA adducts, sperm are selected from either untreated semen samples (12) or semen treated by a swim-up (3,5) or DGC (13) procedure. To our knowledge, no study has been conducted to compare BPDE-DNA adduct levels in selected and nonselected sperm.…”