The main goal of this study was to investigate whether and at what level damage of paternal DNA influences fertilization of oocytes and early embryonic development. We hypothesized that posttesticular sperm DNA damage will only marginally affect sperm physiology due to the lack of gene expression, but that it will affect embryo development at the stage that embryo genome (including the paternal damaged DNA) expression is initiated. To test this, we artificially induced sperm DNA damage by irradiation with x- or gamma rays (doses of 0-300 Gy). Remarkably, sperm cells survived the irradiation quite well and, when compared with nonirradiated cells, sperm motility and integrity of plasma membrane, acrosome, and mitochondria were not altered by this irradiation treatment. In contrast, a highly significant logarithmic relation between irradiation dose and induced DNA damage to sperm cells was found by both terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and the acridin orange assay. Despite the DNA damage, irradiated sperm cells did not show any sign of apoptosis (nuclear fragmentation, depolarization of inner mitochondrial membranes, or phospholipid scrambling) and were normally capable of fertilizing oocytes, as there was no reduction in cleavage rates when compared with nonirradiated sperm samples up to irradiation doses of less than 10 Gy. Further embryonic development was completely blocked as the blastocyst rates at days 7 and 9 dropped from 28% (nonirradiated sperm) to less than 3% by greater than 2.5-Gy-irradiated sperm. This block in embryonic development was accompanied with the initiation of apoptosis after the second or third cleavage. Specific signs of apoptosis, such as nuclear fragmentation and aberrations in spindle formation, were observed in all embryos resulting from in vitro fertilization with irradiated sperm (irradiation doses >1.25 Gy). The results show that sperm DNA damage does not impair fertilization of the oocyte or completion of the first 2-3 cleavages, but blocks blastocyst formation by inducing apoptosis. Embryos produced by assisted reproductive techniques (ART) could have incorporated aberrant paternal DNA (frequently detected in sperm of sub/infertile males). Analogously, in the present work, we discuss the possibility of following embryo development of oocytes fertilized by ART through the blastocyst stage before embryo transfer into the uterus in order to reduce risks of reproductive failure.

https://www.onlinelibrary.wiley.com/doi/pdf/10.2164/jandrol.04152

DNA damage in bovine sperm does not block fertilization and early embryonic development but induces apoptosis after the first cleavages.

To examine whether biomarkers such as sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) can predict cancer development, a nested case-control study was performed in a blackfoot endemic area with a known high cancer risk. A cohort of 686 residents was recruited from three villages in the blackfoot endemic area. Personal characteristics were collected, and venous blood was drawn for lymphocyte culture and stored in a refrigerator. The vital status and cancer development were followed using the National Death Registry, Cancer Registry, and Blackfoot Disease Registry. The follow-up period was from August 1991 to July 1995. During this 4-year period, 31 residents developed various types of cancer. Blood culture samples from nine of these subjects were unsuitable for experiments due to improper storage. Finally, a total of 22 cancer cases had cytogenetic samples that could be analyzed. Twenty-two control subjects were selected from those who did not develop cancer in the study period, and these subjects were matched to cases by sex, age, smoking habits, and residential area. The results showed that there was no significant difference in the frequencies of SCE and chromatid-type CAs between the case and control groups. However, the frequencies of chromosome-type CAs, e.g., chromosome-type gaps, chromosome-type breaks, chromosome-type breaks plus exchanges, total chromosome-type aberrations, and total frequencies of CAs in the case group, were significantly higher than those in the control group (P 1.007% were 11.0 and 12.0, respectively (P 4.023% had a 9-fold increase for cancer risk. These results indicate that chromosome-type CAs are good biomarkers for the prediction of cancer development, whereas SCEs and chromatid-type CAs cannot predict cancer risk.

https://cancerres.aacrjournals.org/content/canres/59/7/1481.full.pdf

Increased chromosome-type chromosome aberration frequencies as biomarkers of cancer risk in a blackfoot endemic area

The alkaline Comet assay was selected as a biomarker of exposure to evaluate the ongoing exposure to ionizing radiation of 50 medical workers occupationally exposed to ionizing radiation and 50 corresponding unexposed control subjects. The primary DNA damage was evaluated by measuring the extent of DNA migration in peripheral blood leukocytes. The inter-individual differences in DNA damage between exposed subjects were compared with their dosimeter readings and occupation. It was found that medical workers who were occupationally exposed to ionizing radiation for different periods of time showed highly significant increases in levels of DNA damage compared with controls. However, influences of the different occupational settings and doses absorbed on the levels of DNA damage, assessed by use of the Comet assay, might be excluded in the majority of subjects. Differences in comet parameters measured due to smoking and gender were not statistically significant in either exposed or control subjects. The results obtained have confirmed the usefulness of the alkaline Comet assay as an additional complement to standard biodosimetric methods. By detection of momentary DNA damage and/or repair activity, it reflects the concurrent exposure and the actual levels of DNA damage present in peripheral blood leukocytes of the radiological workers at the moment of blood sampling.

/pdf/the-alkaline-comet-assay-as-biomarker-in-assessment-of-dna-1x0tvhf9jf.pdf

The alkaline Comet assay as biomarker in assessment of DNA damage in medical personnel occupationally exposed to ionizing radiation

To assess the risks of early life exposure to environmental tobacco smoke (ETS), we tested whether four biomarkers in peripheral blood were associated with home ETS exposure in Hispanic and African-American children. The biomarkers included cotinine (a metabolite of nicotine) and three indicators of molecular and genetic damage from mutagens/carcinogens, protein adducts formed by the carcinogens 4-aminobiphenyl (4-ABP) and polycyclic aromatic hydrocarbons (PAHs), and sister chromatid exchanges (SCEs). We also explored possible ethnic differences in biomarkers. The study cohort comprised 109 Hispanic and African-American preschool children (1–6 years of age). Plasma cotinine was analyzed by gas chromatography, 4-ABP-hemoglobin adducts by gas chromatography-mass spectroscopy, PAH-albumin adducts by ELISA, and SCEs by cytogenetic techniques. Data on the amount of smoking by mothers (average 10.5 cigarettes per day) and other household members and regular visitors (average 6.5 cigarettes per day) were obtained by interview-administered questionnaires. Cotinine, 4-ABP-hemoglobin adducts, and PAH-albumin were significantly higher ( P < 0.05) in the ETS-exposed children compared with the unexposed. SCEs were marginally higher ( P = 0.076). African–American children had higher levels of cotinine ( P = 0.059) and PAH-albumin ( P = 0.02) than Hispanic children, after controlling for exposure to ETS. These results indicate molecular and genetic damage in minority children with relatively low exposure to ETS. They highlight the need for smoking prevention and cessation programs in women of reproductive age and in families with young children.

https://cebp.aacrjournals.org/content/cebp/8/5/427.full.pdf

Molecular and genetic damage from environmental tobacco smoke in young children.

The validity of the micronucleus test as a biomarker of chromosome damage in dividing mammalian cells is well established. This assay was used to study the response of peripheral lymphocytes of a 34-yr-old male patient following treatment with 131I ablative radiation therapy following a total thyroidectomy. Coincidentally, 8 mo before diagnosis, the patient had provided a blood sample for an in vitro study of micronucleus induction following exposure to graded doses of x-rays. The background frequency in the unexposed culture showed a mean count of 6.0 micronuclei per 1000 binucleated (first division) lymphocytes, while mean values of 18.5, 29.0, 41.0, 61.0 and 75.5 micronuclei/1000 cells were observed following x-ray doses of 5, 10, 15, 20, and 25 cGy, respectively. These data fit a nonthreshold, linear dose-response function (y = 2.78x + 3.71; r = .99). Eight months after the in vitro x-ray study, the subject was diagnosed with thyroid cancer. Surgery was performed, and 5 wk later the patient received 1.78 GBq (48 mCi) of 131I as adjuvant radiation therapy. Blood was drawn 11 d after the radiation treatment and at monthly intervals thereafter to analyze the frequency and persistence of micronuclei. The first posttreatment sample showed 35.5 micronuclei per 1000 binucleate cells. Based on the linear dose-response equation from the earlier study, the sixfold increase in micronucleus frequency suggests a dose to the peripheral blood of approximately 11 cGy. The cytogenetic dose estimate compares to approximately 30 cGy using a new model based on external whole-body counting data. Nine consecutive monthly samples have been analyzed to date. Although the micronucleus count has fluctuated (four- to sixfold above background), the frequency after 8 mo is equivalent to the first posttreatment sample. Data show that radiation-induced cellular lesions persist for months following relatively brief radiation exposure to a medical isotope. Results of this study support the conclusion that the lymphocyte micronucleus test is a rapid, sensitive, and perhaps quantitative biomarker of low-dose (< 25 cGy) radiation exposure.

Effect of in vivo exposure to iodine‐131 on the frequency and persistence of micronuclei in human lymphocytes

Structural changes in chromosomes were one of the first recognized forms of genetic injury resulting from exposure to physical and chemical agents. Depending on the lesions induced in the DNA, and therefore, on the nature of the genotoxic substances, damage to chromosomes falls into two categories, structural aberrations and sister chromatid exchange (SCE). Because these two end points respond differently to chromatin lesions, they are complementary in any study designed to identify potential exposure. The circulating lymphocyte in the human is an appropriate cell type in which to measure cytogenetic changes because it is readily accessible, carries genotoxic substances and their metabolites throughout the body, is long-lived, and can integrate exposure. In animal and human studies, it has been shown that both SCEs and aberrations can persist in the lymphocyte following acute or chronic exposure.

Anthony V. Carrano

Papers

Chromosomal alterations as markers of exposure and effect.