Khaled Habas

Development of an in vitro test system for assessment of male, reproductive toxicity

There is a need for improved reproductive toxicology assays that do not require large numbers of animals but are sensitive and informative. Therefore, Staput velocity-sedimentation separation followed by culture of specific mouse testicular cells was used as such a system. The specificity of separation was assessed using immunocytochemistry to identify spermatids, spermatocytes and spermatogonia. The efficacy of the system to detect toxicity was then evaluated by analysing the effects of hydrogen peroxide (H2O2) by the terminal uridine-deoxynucleotide end-labelling (TUNEL) assay to show the rate of apoptosis induced among the different types of germ cells. We found that 2 h of treatment at both 1 and 10 μM induced increases of over ∼10-fold in the percentage of apoptotic cells (p≤0.001), confirming that testicular germ cells are prone to apoptosis at very low concentrations of H2O2. It was also demonstrated for the first time for this compound that spermatogonia are significantly more susceptible than spermatocytes, which are more affected than spermatids. This reflects the proportion of actively dividing cells in these cell types, suggesting a mechanism for the differential sensitivity. The approach should thus form the basis of a useful test system for reproductive and genetic toxicology in the future.

Germ Cell Responses to Doxorubicin Exposure in Vitro

Anthracyclines such as doxorubicin (Dox), widely used to treat various types of tumours, may result in induced testicular toxicity and oxidative stress. The present investigation was designed to determine whether exposure of isolated and purified mouse germ cells to Dox induces DNA damage in the form of strand breaks (presumably) resulting in apoptosis and to investigate the relative sensitivity of specific cell types. DNA damage was assessed using the Comet assay and the presence of apoptosis was determined by TUNEL assay. Isolated mouse germ cells were treated with different concentrations (0.05, 0.5 and 1mM, respectively) of Dox, and fixed 1h after treatment. The incidences of both DNA damage shown by single cell gel-electrophoresis and of apoptosis increased significantly in each specific cell type in a concentration-dependent manner. The DNA damage and apoptosis incidences gradually increased with concentration from 0.05 to 1mM with Dox. Our results indicate that apoptosis plays a vital role in the induction of germ cell phase-specific toxicity caused by Dox with pre-meiotically and meiotically dividing spermatogonia and spermatocytes respectively as highly susceptible target cells.

Diethylstilbestrol induces oxidative DNA damage, resulting in apoptosis of spermatogonial stem cells in vitro

The spermatogonial stem cells (SSCs) are the only germline stem cells in adults that are responsible for the transmission of genetic information from mammals to the next generation. SSCs play a very important role in the maintenance of progression of spermatogenesis and help provide an understanding of the reproductive biology of future gametes and a strategy for diagnosis and treatment of infertility and male reproductive toxicity. Androgens/oestrogens are very important for the suitable maintenance of male germ cells. There is also evidence confirming the damaging effects of oestrogen-like compounds on male reproductive health. We investigated the effects in vitro, of diethylstilbestrol (DES) on mouse spermatogonial stem cells separated using Staput unit-gravity velocity sedimentation, evaluating any DNA damage using the Comet assay and apoptotic cells in the TUNEL assay. Immunocytochemistry assays showed that the purity of isolated mouse spermatogonial cells was 90%, and the viability of these isolated cells was over 96%. Intracellular superoxide anion production (O2-) in SSCs was detected using p-Nitro Blue Tetrazolium (NBT) assay. The viability of cells after DES treatment was examined in the CCK8 (cell counting kit-8) cytotoxicity assay. The results showed that DES-induced DNA damage causes an increase in intracellular superoxide anions which are reduced by the flavonoid, quercetin. Investigating the molecular mechanisms and biology of SSCs provides a better understanding of spermatogonial stem cell regulation in the testis.

In vitro responses to known in vivo genotoxic agents in mouse germ cells

Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n‐ethyl‐n‐nitrosourea (ENU), 6‐mercaptopurine (6‐MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration‐dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration‐related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6‐MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real‐time PCR results showed that the mRNA level expression of p53 increased and bcl‐2 decreased significantly with the increasing ENU, 6‐MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6‐MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6‐MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro. Environ. Mol. Mutagen. 58:99–107, 2017.

Silver nanoparticle-mediated cellular responses in isolated primary Sertoli cells in vitro

The present study explored the mechanism of cytotoxic and genotoxic effects of AgNPs on a primary culture of mouse Sertoli cells in vitro. To understand the possible molecular mechanisms of testicular lesions following exposure to AgNPs, isolated Sertoli cells were exposed to 5, 10, or 15 μg/ml. DNA damage in the Comet assay and apoptosis in the TUNEL assay were evaluated. The mRNA expression of p53 and bcl-2 genes and their proteins involved in apoptosis was also investigated. The antioxidant status of treated Sertoli cells was determined by measuring superoxide dismutase (SOD-1), catalase (CAT) and glutathione peroxidase (GPX-1) using quantitative polymerase chain reaction (qPCR). The superoxide anions were detected using the nitroblue tetrazolium (NBT) reduction assay. Results indicated that AgNP exposure causes increased oxidative stress levels. The activation of p53, repression of bcl-2 and reduction of endogenous antioxidant enzymes were also involved in these mechanistic pathways, leading to reduced cell numbers and cell detachment.

DNA damage protection by bulk and nano forms of quercetin in lymphocytes of patients with chronic obstructive pulmonary disease exposed to the food mutagen 2-amino-3-methylimidazo [4,5-f]quinolone (IQ)

ABSTRACT Chronic obstructive pulmonary disease (COPD) in humans, describes a group of lung conditions characterised by airflow limitation that is poorly reversible. The airflow limitation usually progresses slowly and is related to an abnormal inflammatory response of the lung to toxic particles. COPD is characterised by oxidative stress and an increased risk of lung carcinoma. The 2‐amino‐3‐methylimidazo [4,5‐f]quinoline (IQ) is one of a number of mutagenic/carcinogenic heterocyclic amines found mainly in well‐cooked meats which are thus part of the regular diet. Antioxidants are very important in order to protect the cells against oxidative damage. The aim of the present study was to assess the effects of IQ on the level of DNA damage and susceptibility to a potent mutagen in peripheral blood cells of COPD patients. DNA damage and the frequency of micronuclei (MNi) were evaluated using the Comet and micronucleus assays, respectively. Differential expressions of both mRNA and protein of the endogenous antioxidant enzyme catalase were evaluated with quantitative polymerase chain reaction (qPCR) and Western blot analysis, respectively. Furthermore, the effect of bulk and nano forms of quercetin and their combination with IQ were examined. Results of the present study clearly demonstrated that MNi frequency in the peripheral blood lymphocytes exhibited a positive correlation with the DNA damage as evident from the different Comet assay parameters. Increase of the endogenous antioxidant catalase also showed there was a stimulation of this enzyme system by IQ. Whereas, the endogenous antioxidant quercetin significantly reduced oxidative stress in COPD patients and healthy individuals. HighlightsIn COPD patients and healthy individuals IQ increased DNA damage and frequency of micronuclei.Induction of catalase indicates a stimulation of the antioxidant enzyme system.Quercetin significantly reduced oxidative stress in COPD patients and healthy individuals.The bulk version of quercetin was compared directly to its nano form which caused greater reduction.