Volume 11 (2018): Edition 1 (May 2018)

Safety assessment of chemicals, pharmaceuticals, food and food ingredients, cosmetics, industrial products is very crucial prior to their approval for human uses. Since the commencement of toxicity testing (about 500 years ago, since 1520), significant advances have been made with respect to the 3Rs (reduction, refinement and replacement) alternative approaches. This review is focused on the update in acute systemic toxicity testing of chemicals. Merits and demerits of these advances were also highlighted. Traditional LD₅₀ test methods are being suspended while new methods are developed and endorsed by the regulatory body. Based on the refinement and reduction approaches, the regulatory body has approved fixed dose procedure (FDP), acute toxic class (ATC) method and up and down procedure (UDP) which involves few numbers of animals. In terms of replacement approach, the regulatory body approved 3T3 neutral red uptake (NRU), the normal human keratinocyte (NHK), and the 3T3 neutral red uptake (NRU) phototoxicity test for acute phototoxicity. However, other promising replacement alternatives such as organ on chip seeded with human cells for acute systemic toxicity and 3T3 neutral red uptake (NRU) cytotoxicity test for identifying substances not requiring classification, as well as the in silico approaches are yet to receive regulatory approval. With this backdrop, a collaborative effort is required from the academia, industries, regulatory agencies, government and scientific organizations to ensure speedily regulatory approval of the prospective alternatives highlighted.

Pixantrone (PIX) is an anticancer drug approved for the treatment of multiple relapsed or refractory aggressive B-cell non-Hodgkin’s lymphoma. It is an aza-anthracenedione synthesized to have the same anticancer activity as its predecessors, anthracyclines (e.g. doxorubicin) and anthracenediones (e.g. mitoxantrone), with lower cardiotoxicity. However, published data regarding its possible cardiotoxicity are scarce. Therefore, this work aimed to assess the potential cytotoxicity of PIX, at clinically relevant concentrations (0.1; 1; and 10 µM) in both non-differentiated and 7-day differentiated H9c2 cells. Cells were exposed to PIX for 48 h and cytotoxicity was evaluated through phase contrast microscopy, Hoescht staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red (NR) uptake assays. Cytotoxicity was observed in differentiated and non-differentiated H9c2 cells, with detached cells and round cells evidenced by phase contrast microscopy, mainly at the highest concentration tested (10 µM). In the Hoechst staining, PIX 10 µM showed a marked decrease in the number of cells when compared to control but with no signs of nuclear condensation. Furthermore, significant concentration-dependent mitochondrial dysfunction was observed through the MTT reduction assay. The NR assay showed similar results to those obtained in the MTT reduction assay in both differentiated and non-differentiated H9c2 cells. The differentiation state of the cells was not crucial to PIX effects, although PIX toxicity was slightly higher in differentiated H9c2 cells. To the best of our knowledge, this was the first in vitro study performed with PIX in H9c2 cells and it discloses worrying cytotoxicity at clinically relevant concentrations.

Toluene abuse is one of the most common addictions among marginalized Roma. The aim of the study was the comparison of urinary toluene metabolite levels in marginalized population of Eastern Slovakia as compared to the majority population, and to verify the validity of the answers, given in the questionnaires, regarding toluene abuse. The study was carried out as part of the HEPA-META project aiming to map the prevalence of health problems in marginalized Roma. The majority of people living outside the area of the segregated Roma communities comprised the control group. The total number of study participants was 770. Statistically significant differences in the levels of hippuric acid and o-cresol were found between Roma and the majority population. Variations in urinary hippuric acid levels in addition to toluene abuse can be caused also by dietary factors, medical treatment as well as alcohol consumption, which is frequent (not only) in marginalized communities.

Alar (Daminozide) is a plant growth regulator which is widely used as a fruit preservative for apple and mango to prevent pre-harvest fruit drop, promote color development and to delay excessive ripening. The aim of the present work was to demonstrate the effect of Alar on several life history traits, adult morphology, Hsp70 protein expression and in vivo DNA damage in the brain of the model organism Drosophila melanogaster. We assessed the life history and morphological traits including fecundity, developmental time, pupation height, egg-to-adult viability and mean wing length, body length, arista length and sternopleural bristle number of the emerging flies. The results showed a significant delay in the developmental milestones, increase in body length, wing length, arista length, a decrease in fecundity, pupal height and variation in sternopleural bristle number in the treated flies in comparison to the controls. Overexpression of Hsp70 protein suggests alar induced subcellular molecular stress and comet assay validates genotoxicity in the form of DNA damage in the treated larvae. Mutation screening experiment revealed induction of X lined lethal mutation.

Thiourea (thiophen-3-yl-acetic acid) is a well established antithyroid drug used for treating hyperactivity of the thyroid gland as it blocks the conversion of thyroxine (T4) to triiodothyronine (T3) in peripheral tissues. Human exposures to thiourea include contaminated drinking water and vegetables for its extensive use in fertilizers. Chronic thiourea exposure can cause thyroid dysfunction leading to redox imbalance. However, such effects on morphological, quantitative, functional and hypothalamo-pituitary-adrenocortical axis (HPA) analysis of the adrenal gland are yet to be explored. The aim was to explore the effect of thiourea on structural and functional status of the adrenocortical region with special reference to the HPA axis. Control rats were fed a normal laboratory standardized diet whereas to experimental rats, thiourea at a dose of 0.3 mg/day/Kg body weight was administered orally, once every day for consecutive 28 days. Histology and histometry, including morphometry of the adrenal, adrenal ∆5 3β HSD and 17β HSD activity, LPO level and serum corticosterone profile were assessed. Statistical significance was studied by ‘Mann-Whitney U’ test at p<0.05. Hypertrophy and hyperplasia of the adrenocortical cells was found especially in the layer zona fasciculata (p=0.0027) and enhanced adrenal ∆5 3β HSD activity (p=0.0067) in comparison to that of the control. Increased lipid peroxidation (p=0.0054) and up-regulated corticosterone release (p=0.0064) through adrenocortical stress signalling pathway were also noted. Stereological analysis of the left adrenal gland showed significant increase in volume (p=0.0025) and mass of cells (p=0.0031) in adrenocortical region in comparison to that of control animals. This study concludes that thiourea, in addition to its antithyroidal activity, develops stress in the adrenal as evident by enhanced lipid peroxidation in the gland that in turn through the HPA axis causes hypertrophy and hyperplasia of adrenocortical cells to enhance synthesis and release of corticosterone secretion to counteract the stress developed under the influence of this potent chemical agent.