Combinations of genotoxic tests for the evaluation of group 1 IARC carcinogens.

Many of the known human carcinogens are potent genotoxins that are efficiently detected as carcinogens in human populations but certain types of compounds such as immunosuppressants, sex hormones, etc. act via non-genotoxic mechanism. The absence of genotoxicity and the diversity of modes of action of non-genotoxic carcinogens make predicting their carcinogenic potential extremely challenging. There is evidence that combinations of different short-term tests provide a better and efficient prediction of human genotoxic and non-genotoxic carcinogens. The purpose of this study is to summarize the in vivo and in vitro comet assay (CMT) results of group 1 carcinogens selected from the International Agency for Research on Cancer and to discuss the utility of the comet assay along with other genotoxic assays such as Ames, in vivo micronucleus (MN), and in vivo chromosomal aberration (CA) test. Of the 62 agents for which valid genotoxic data were available, 38 of 61 (62.3%) were Ames test positive, 42 of 60 (70%) were in vivo MN test positive and 36 of 45 (80%) were positive for the in vivo CA test. Higher sensitivity was seen in in vivo CMT (90%) and in vitro CMT (86.9%) assay. Combination of two tests has greater sensitivity than individual tests: in vivo MN + in vivo CA (88.6%); in vivo MN + in vivo CMT (92.5%); and in vivo MN + in vitro CMT (95.6%). Combinations of in vivo or in vitro CMT with other tests provided better sensitivity. In vivo CMT in combination with in vivo CA provided the highest sensitivity (96.7%).

Evaluation of the impact of bioaccumulation of PAH from the marine environment on DNA integrity and oxidative stress in marine rock oyster (Saccostrea cucullata) along the Arabian sea coast.

Marine pollution due to oil spills is of great concern globally for their impact on the health of marine ecosystems. We assessed the genotoxic effects and oxidative stress due to genotoxic pollutants accumulated from the ambient marine environment in the tissues of marine rock oyster, Saccostrea cucullata along the Arabian Sea coast around Goa, India. The extent of DNA damage in S. cucullata was determined by comet assay as variation of comet parameter: mean % tail DNA along the coast with respect to that at the reference site (Tiracol, Goa, India). In addition, the oxidative stress responses of rock oysters exposed to marine pollutants such as polycyclic aromatic hydrocarbons (PAHs) were assessed as a function of variation in antioxidant enzyme activities such as glutathione-s-transferase (GST), catalase (CAT) and superoxide dismutase (SOD) along the coast. Spearman correlation analysis showed significant correlation between different components of PAHs (viz., 2-3-PAH, 4-6-PAH and oxy-PAH) in the tissues of the rock oysters and the antioxidant enzyme activities. The antioxidant enzyme activities in S. cucullata increased with increasing concentrations of PAHs in tissues in the following order of sampling sites: Tiracol < Arambol < Betul < Velsao. Among the PAHs, oxy-PAH was found to be most predominant in causing DNA damage in S. cucullata. These results provide an insight into environmental genotoxicity and oxidative stress induced by PAHs along the Arabian Sea coast, India.

Genotoxicity of two heavy metal compounds: lead nitrate and cobalt chloride in Polychaete Perinereis cultrifera.

The present study explores the in vivo and in vitro genotoxic effects of lead nitrate, [Pb(NO3)2] a recognized environmental pollutant and cobalt chloride (CoCl2), an emerging environmental pollutant in polychaete Perinereis cultrifera using comet assay. Despite widespread occurrence and extensive industrial applications, no previous published reports on genotoxicity of these compounds are available in polychaete as detected by comet assay. Polychaetes were exposed in vivo to Pb(NO3)2 (0, 100, 500, and 1000 µg/l) and CoCl2 (0, 100, 300, and 500 µg/l) for 5 days. At 100 µg/l Pb(NO3)2 concentration, tail DNA (TDNA) values in coelomocytes were increase by 1.16, 1.43, and 1.55-fold after day 1, day 3, and day 5, whereas, OTM showed 1.12, 2.33, and 2.10-fold increase in in vivo. Pb(NO3)2 showed a concentration and time-dependent genotoxicity whereas CoCl2 showed a concentration-dependent genotoxicity in in vivo. A concentration-dependent increase in DNA damage was observed in in vitro studies for Pb(NO3)2 and CoCl2. DNA damage at 500 µg/L showed almost threefold increase in TDNA and approximately fourfold increase in OTM as compared to control in in vitro. Our studies suggest that Pb(NO3)2 and CoCl2 have potential to cause genotoxic damage, with Pb(NO3)2 being more genotoxic in polychaete and should be used more carefully in industrial and other activities. Graphical abstract.

Genotoxicity of cadmium chloride in the marine gastropod Nerita chamaeleon using comet assay and alkaline unwinding assay.

This paper presents an evaluation of the genotoxic effects of cadmium chloride (CdCl2 ) on marine gastropod, Nerita chamaeleon following the technique of comet assay and the DNA alkaline unwinding assay (DAUA). In this study, the extent of DNA damage in gill cells of N. chamaeleon was measured after in vivo exposure to four different concentrations (10, 25, 50, and 75 µg/L) of CdCl2 . In vitro exposure of hydrogen peroxide (H2 O2 ; 1, 10, 25, and 50 µM) of the gill cells showed a significant increase in the percentage tail DNA, Olive tail moment, and tail length (TL). Significant changes in percentage tail DNA by CdCl2 exposure were observed in all exposed groups of snails with respect to those in control. Exposure to 75 µg/L of CdCl2 produced significant decrease in DNA integrity as measured by DAUA at all duration with respect to control. In vivo exposure to different concentrations of CdCl2 (10, 25, 50, and 75 µg/L) to N. chamaeleon showed considerable increase in DNA damage as observed by both alkaline comet assay and the DAUA. The extent of DNA damage in marine gastropods determined by the application of alkaline comet assay and DAUA clearly indicated the genotoxic responses of marine gastropod, N. chamaeleon to a wide range of cadmium concentration in the marine environment.

Evaluation of impairment of DNA in marine gastropod, Morula granulata as a biomarker of marine pollution.

The impairment of DNA in marine gastropod Morula granulata was evaluated in terms of the loss of DNA integrity in the species as a measure of the impact of genotoxic contaminants prevalent in the marine environment along the coast of Goa, India. The extent of DNA damage occurred in the marine gastropods collected from different sampling sites such as Arambol, Anjuna, Sinquerim, Dona Paula, Bogmalo, Hollant, Velsao, Betul and Palolem along the coast of Goa was measured following the technique of partial alkaline unwinding as well as comet assays. The highest DNA integrity was observed at Arambol (F, 0.75), identified as the reference site, whereas the lowest DNA integrity at Hollant (F, 0.33) situated between the two most contaminated sites at Bogmalo and Velsao. The impact of genotoxic contaminants on marine gastropods was pronounced by their low DNA integrity at Sinquerim (F, 0.40) followed by Betul (F, 0.47), Velsao (F, 0.51), Anjuna (F, 0.54), Bogmalo (F, 0.55), Dona Paula (F, 0.67) and Palolem (F, 0.70). The extent of DNA damage occurred in M. granulata due to ecotoxicological impact of the prevailing marine pollutants along the coast of Goa was further substantiated by comet assay and expressed in terms of %head-DNA, %tail DNA, tail length and Olive tail moment. The single cell gel electrophoresis of M. granulata clearly showed relatively higher olive tail moment in the marine gastropod from the contaminated sites, Anjuna, Hollant, Velsao and Betul. The variation in the mean %head DNA at different sampling sites clearly indicated that the extent of DNA damage in marine gastropod increases with the increase in the levels of contamination at different sampling sites along the coast. The stepwise multiple regression analysis of the water quality parameters showed significant correlation between the variation in DNA integrity and PAH in combination with NO3, salinity and PO4 (R¯(2), 0.90). The measurement of DNA integrity in M. granulata thus provides an early warning signal of contamination of the coastal ecosystem of Goa by genotoxic contaminants.

Southeast Asia's environmental challenges: emergence of new contaminants and advancements in testing methods.

Emerging contaminants, including pharmaceuticals, personal care products, microplastics, and per- and poly-fluoroalkyl substances, pose a major threat to both ecosystems and human health in Southeast Asia. As this region undergoes rapid industrialization and urbanization, the increasing presence of unconventional pollutants in water bodies, soil, and various organisms has become an alarming concern. This review comprehensively examines the environmental challenges posed by emerging contaminants in Southeast Asia and recent progress in toxicity testing methods. We discuss the diverse range of emerging contaminants found in Southeast Asia, shedding light on their causes and effects on ecosystems, and emphasize the need for robust toxicological testing methods. This review is a valuable resource for researchers, policymakers, and environmental practitioners working to mitigate the impacts of emerging contaminants and secure a sustainable future for Southeast Asia.

Combined exposure to nanoplastics and metal oxide nanoparticles inhibits efflux pumps and causes oxidative stress in zebrafish embryos.

The ubiquity of microplastic/nanoplastics (MP/NPs) provides an opportunity for their interaction with other widely spread environmental contaminants. MP/NP and nanoparticles share a similar transport route from sources, production, and disposal. Metal oxide nanoparticles (nMOx) have varied industrial applications, and limited knowledge is available on their interaction with MP/NPs. The present study investigated the effect of NPs (1 mg/L) on the efflux of two nMOx, aluminium oxide nanoparticles (nAl2O3, 1 mg/L) and cerium oxide nanoparticles (nCeO2, 1 mg/L), and their combined toxicity to zebrafish embryos. The results illustrated increased accumulation of aluminium and cerium in the combined exposure group compared to the nMOx alone treatment. The presence of NPs exacerbated the oxidative stress caused by nAl2O3 and nCeO2, as evidenced by an increase in the concentration of reactive oxygen species (ROS), alteration of antioxidants, and lipid peroxidation. The integrated biomarker response (IBRv2) values showed the induction of an antioxidative response in NP + nAl2O3, whereas a decline in IBRv2 values was observed in NP + nCeO2. Our results indicate that NPs aggravated the accumulation of nMOx and their toxicity. The present work highlights that more attention should be paid to the discharge of these contaminants into the natural environment.

A comprehensive review on environmental toxicity of azole compounds to fish.

BACKGROUND: Azoles are considered as one of the most efficient fungicides for the treatment of humans, animals, and plant fungal pathogens. They are of significant clinical importance as antifungal drugs and are widely used in personal care products, ultraviolet stabilizers, and in aircraft for its anti-corrosive properties. The prevalence of azole compounds in the natural environment and its accumulation in fish raises questions about its impact on aquatic organisms. OBJECTIVES: The objective of this paper is to review the scientific studies on the effects of azole compounds in fish and to discuss future opportunities for the risk evaluation. METHODS: A systematic literature search was conducted on Web of Science, PubMed, and ScienceDirect to locate peer-reviewed scientific articles on occurrence, environmental fate, and toxicological impact of azole fungicides on fish. RESULTS: Studies included in this review provide ample evidence that azole compounds are not only commonly detected in the natural environment but also cause several detrimental effects on fish. Future studies with environmentally relevant concentrations of azole alone or in combination with other commonly occurring contaminants in a multigenerational study could provide a better understanding. CONCLUSION: Based on current knowledge and studies reporting adverse biological effects of azole on fish, considerable attention is required for better management and effective ecological risk assessment of these emerging contaminants.

Toxicological interactions of microplastics/nanoplastics and environmental contaminants: Current knowledge and future perspectives.

The co-occurrence of microplastics/nanoplastics (MPs/NPs) with other environmental contaminants has stimulated a focus shift of its skyrocketed research publications (more than 3000 papers during 2016-2020, Web of Science) from ubiquitous occurrence to interactive toxicity. Here, in this review, we provided the current state of knowledge on toxicological interaction of MPs/NPs with co-contaminants (heavy metals, polycyclic aromatic hydrocarbons, pharmaceuticals, pesticides, nanoparticles, organohalogens, plastic additives, and organotins). We discussed the possible interactions (aggregation, adsorption, accumulation, transformation, desorption) that played a role in influencing the toxicity of the mixture. Besides, the type of interactions such as synergistic, antagonistic, potentiating was expounded to get a deeper mechanistic understanding. Despite the wide occurrence and usage, scant studies were available on polypropylene, polyethylene terephthalate. Our analysis shows a dearth of research on common occurring heavy metals (mercury, lead, chromium), phthalates, personal care products. Considerations for environmental factors such as the presence of dissolved organic matter, pH, salinity, temperature, and effects of different colors and types of polymer are recommended.

Effects of nanoplastic on toxicity of azole fungicides (ketoconazole and fluconazole) in zebrafish embryos.

The ubiquity of nanoplastics (NPs) raises concerns about their interactions and combined toxicity with other common contaminants. Although azoles are present throughout the natural environment, their interactions with NP are not well known. We investigated the effects of polystyrene (PS) NP on the toxicity of ketoconazole (KCZ) and fluconazole (FCZ) in zebrafish embryos using the developmental toxicity, oxidative-stress-related biochemical parameters, and expression of genes related to neurotoxicity (ache), cardiotoxicity (gata4, bmp4), inflammation (il1b), oxidative stress (sod1, sod2, cyp1a), and apoptosis (bax, bcl2). Co-exposure to NP (1 mg/L) and KCZ/FCZ (1 mg/L) for 96 h reduced the hatching rate, survival rate, and heart rate and increased the malformation rate and catalase activity. The bax/bcl2 ratio, an apoptosis indicator, was higher after NP, KCZ, or FCZ treatment. However, the bax/bcl2 ratio after exposure to NP + KCZ or NP + FCZ was much higher than that after single exposure. Overall, the results indicated that NP aggravated the toxicity of azole by significantly increasing the reactive oxygen species, lipid peroxidation and altering the expression of oxidative-stress- and apoptosis-related genes. The interactive toxicity of PS NP with KCZ/FCZ reported in this study emphasises the need for caution in the release of azole fungicides in the environment.

Metal oxide nanoparticles and polycyclic aromatic hydrocarbons alter nanoplastic's stability and toxicity to zebrafish.

Co-occurrence of nanoplastics (NPs) with metal oxide nanoparticles (nMOx) and polycyclic aromatic hydrocarbons (PAHs) have been widely reported. However, there is a scarcity of information on their interactions and combined toxic effects. In this study, we used two different sized NPs [55 nm (NP1) and 100 nm (NP2)] to understand the effect of nMOx (nCuO and nZnO) and PAHs [chrysene (Chr) and fluoranthene (Flu)] on NPs' stability and toxicity to zebrafish. Results revealed that increasing the concentration of nMOx, zeta-potential increased, and charge reversal was observed in NPs suspension while PAH produced no major changes. Aggregation kinetics performed with nMOx exhibited higher aggregation of NPs in presence of NaCl that alleviated critical coagulation concentration. NP1 stabilized the size of otherwise unstable nMOx suspension in the tap-water for a longer period, whereas, aggregation was observed with NP2. The in vivo comet assay results showed that NP1 was more genotoxic than NP2 owing to their lower size. Interestingly the DNA damage was highest in NPs+nMOx followed by nMOx and NPs. Unlike nMOx, Chr/Flu+NPs showed reduced DNA damage as compared to NPs or PAH alone. Alteration in catalase activity and lipid peroxidation value indicated oxidative stress in all exposure groups.

10-Gingerol Suppresses Osteoclastogenesis in RAW264.7 Cells and Zebrafish Osteoporotic Scales.

Osteoporosis is the most common aging-associated bone disease and is caused by hyperactivation of osteoclastic activity. We previously reported that the hexane extract of ginger rhizome [ginger hexane extract (GHE)] could suppress receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. However, the anti-osteoclastic components in GHE have not yet been identified. In this study, we separated GHE into several fractions using silica gel column chromatography and evaluated their effects on osteoclastogenesis using a RAW264.7 cell osteoclast differentiation assay (in vitro) and the zebrafish scale model of osteoporosis (in vivo). We identified that the fractions containing 10-gingerol suppressed osteoclastogenesis in RAW264.7 cells detected by tartrate-resistant acid phosphatase (TRAP) staining. In zebrafish, GHE and 10-gingerol suppressed osteoclastogenesis in prednisolone-induced osteoporosis regenerated scales to promote normal regeneration. Gene expression analysis revealed that 10-gingerol suppressed osteoclast markers in RAW264.7 cells [osteoclast-associated immunoglobulin-like receptor, dendrocyte-expressed seven transmembrane protein, and matrix metallopeptidase-9 (Mmp9)] and zebrafish scales [osteoclast-specific cathepsin K (CTSK), mmp2, and mmp9]. Interestingly, nuclear factor of activated T-cells cytoplasmic 1, a master transcription regulator of osteoclast differentiation upstream of the osteoclastic activators, was downregulated in zebrafish scales but showed no alteration in RAW264.7 cells. In addition, 10-gingerol inhibited CTSK activity under cell-free conditions. This is the first study, to our knowledge, that has found that 10-gingerol in GHE could suppress osteoclastic activity in both in vitro and in vivo conditions.

Zebrafish: An emerging model to study microplastic and nanoplastic toxicity.

Microplastics (MPs) and nanoplastics (NPs) have received global concern due to its widespread contamination, ingestion in aquatic organisms and the ability to cross the biological barrier. However, our understanding of its bioaccumulation, toxicity, and interaction with other environmental pollutants is limited. Zebrafish is increasingly used to study the bioaccumulation and toxicity of environmental contaminants because of their small size, ease of breed, short life cycle and inexpensive maintenance. The transparent nature of zebrafish embryo and larvae provides excellent experimental advantages over other model organisms in studying the localization of fluorescent-labeled MPs/NPs particles. Zebrafish outplays the traditional rodent models with the availability of transgenic lines, high-throughput sequencing and genetic similarities to humans. All these characteristics provide an unprecedented opportunity to investigate the toxicity of MPs/NPs and associated contaminants. This review summarizes the existing literature on MPs/NPs research in zebrafish and suggests a path forward for future research.