A 2-step remote TUNEL approach for sperm DNA fragmentation assessment. Analysis in donors and patients.

OBJECTIVES: Infertility is a disease of the male or female reproductive systems. Male reproductive workup is based on routine semen analysis, although of limited value. The 2021 WHO Manual incorporated Sperm DNA Fragmentation (SDF) assessment, and highlighted the need for individual laboratories to define suitable thresholds. This study aimed to present an alternative to address this issue, determine an SDF cut-off value with fertile donors, and characterize SDF in a patient cohort and their relationship with semen parameters. STUDY DESIGN: A service unit was established to remotely perform TUNEL assay in a 2 step-process. Semen samples were received at andrology laboratories, subjected to routine semen analysis (WHO, 2010), partially processed and transported to the service unit for SDF evaluation. Using this setting, studies were done in fertile donors (n = 15) to define the cut-off value, and in men undergoing infertility workup (n = 318). RESULTS: A cut-off value of 9.17 % was determined with the fertile donor cohort. With this cut-off, a 64.46 % abnormal SDF incidence was determined in the patient cohort. SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05). TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05). A similar abnormal SDF incidence was determined among patients with semen abnormalities. Asthenozoospermic and ≥40 years patient samples depicted higher (P < 0.05) SDF than those of the general population. SDF incidence was also high in normozoospermic patients. CONCLUSIONS: Using a 2-step remote approach with a standardized procedure and an SDF cut-off value established with fertile donors, high SDF incidence in semen samples depicting normal and abnormal quality were identified in men consulting for infertility, highlighting the relevance of its evaluation as part of the male fertility workup.

Assessment of mutations induced by cold atmospheric plasma jet treatment relative to known mutagens in Escherichia coli.

The main bactericidal components of cold atmospheric plasma (CAP) are thought to be reactive oxygen and nitrogen species (RONS) and UV-radiation, both of which have the capacity to cause DNA damage and mutations. Here, the mutagenic effects of CAP on Escherichia coli were assessed in comparison to X- and UV-irradiation. DNA damage and mutagenesis were screened for using a diffusion-based DNA fragmentation assay and modified Ames test, respectively. Mutant colonies obtained from the latter were quantitated and sequenced. CAP was found to elicit a similar mutation spectrum to X-irradiation, which did not resemble that for UV implying that CAP-produced RONS are more likely the mutagenic component of CAP. CAP treatment was also shown to promote resistance to the antibiotic ciprofloxacin. Our data suggest that CAP treatment has mutagenic effects that may have important phenotypic consequences.

Toxicological assessment of Opuntia dillenii (Ker Gawl.) Haw. cladode methanol extract, fractions and its alpha pyrones: Opuntiol and opuntioside.

ETHNOPHARMACOLOGICAL RELEVANCE: The edible plant Opuntia dillenii (Ker Gawl.) Haw. commonly known as Nagphana, belongs to the Cactaceae family. It is traditionally used to treat various ailments including inflammation, gastric ulcers, diabetes, hepatitis, asthma, whooping cough and intestinal spasm. AIM OF THE STUDY: Despite its traditional use in various countries, detailed toxicological studies of O. dillenii cladode are few. Thus in the current study, toxicity of O. dillenii cladode derived methanol extract, fractions and its α-pyrones: opuntiol and opuntioside have been addressed. METHODS: The test agents were assessed using both in vitro and in vivo toxicity assays. MTT on human embryonic kidney cell line (HEK-293), tryphan blue exclusion in rat neutrophils, Cytokinesis-B block micronucleus (CBMN) in human lymphocytes and genomic DNA fragmentation using agarose gel electrophoresis were performed. In acute toxicity test, mice orally received extract (5 g/kg) for 7 days followed by measurements of relative organ weight, biochemical (blood profile, liver and kidney function test) and histological studies (liver and kidney) were carried out. Rat bone marrow micronucleus genotoxicity assay was also conducted. RESULTS: O. dillenii derived test agents were non-cytotoxic and had no effect on the integrity of DNA. Methanol extract (5 g/kg) orally administered in mice did not cause any significant change in relative organ weights, biochemical parameters and liver and kidney histology as compared to vehicle control. In parallel, extract did not stimulate micronuclei formation in rat bone marrow polychromatic erythrocytes. CONCLUSION: These results led to conclude that edible O. dillenii extract is non-toxic via the oral route and appears to be non-cyto-, hepato-, nephro- or genotoxic, thereby supporting its safe traditional use against various ailments. Therefore, opuntiol and opuntioside may serve as lead compounds in designing new drug(s) derived from edible plants.

In Vitro Apoptotic Cell Death Assessment.

Chemical compounds induce cytotoxicity by various mechanisms, including interference in membrane integrity, metabolism, cellular component degradation or release, and cell division. Between the classic death pathways, namely, autophagy, apoptosis, and necrosis, apoptosis have been in the focus for the last several years as an important pathway for the toxicity of different types of xenobiotics. Because of that, having the tools to evaluate it is key for understanding and explaining the toxicodynamics of different classes of substances. Here, we describe a wide array of classic assays that can be easily implemented to evaluate apoptosis induction.

Assessment of heavy metals and its impact on DNA fragmentation in different fish species / Avaliação de metais pesados e seu impacto na fragmentação de DNA em diferentes espécies de peixe

Abstract This study was conducted to assess water pollution by examining DNA fragmentation in selected fish organs (kidney, liver, gills, and muscle tissue) from Wallago attu, Sperata sarwari, Vulgaris vulgaris, and Labeo rohita collected from a known polluted section of the Chenab River, Pakistan, and from a control site. The fish were caught using a gill net and were assigned to three different weight groups (W1, W2, and W3) to study the degree of variation in DNA fragmentation in relation to body weight. In fish from the polluted site, DNA fragmentation was higher in kidney, liver, gills, and muscles, compared to the control. No significant DNA fragmentation was observed in fish collected from the control site. Highly significant (P < 0.01) relationship between body weight and DNA fragmentation was found in the organs of fish procured at the contaminated site. DNA fragmentation in body organs was found to be affected by the concentrations of lead, copper, nickel, and cadmium in W. attu, S. sarwari, L. rohita, and V. vulgarus harvested from Chenab River. DNA fragmentation in different freshwater fish species is therefore a reliable biomarker of water pollution.

Resumo Este estudo foi conduzido para avaliar a poluição da água examinando a fragmentação do DNA em órgãos de peixes selecionados (rim, fígado, brânquias e tecido muscular) de Wallago attu, Sperata sarwari, Vulgaris vulgaris e Labeo rohita coletados de uma conhecida área poluída do rio Chenab, Paquistão e de um local de controle. Os peixes foram capturados usando uma rede branquial e foram divididos em três grupos de pesos diferentes (W1, W2 e W3) para estudar o grau de variação na fragmentação do DNA em relação ao peso corporal. Nos peixes do local poluído, a fragmentação do DNA foi maior nos rins, fígado, brânquias e músculos, em comparação ao controle. Não foi observada fragmentação significativa do DNA em peixes coletados no local de controle. Relação altamente significativa (P <0,01) entre o peso corporal e a fragmentação do DNA foi encontrada nos órgãos dos peixes adquiridos no local contaminado. Verificou-se que a fragmentação do DNA nos órgãos do corpo é afetada pelas concentrações de chumbo, cobre, níquel e cádmio em W. attu, S. sarwari, L. rohita e V. vulgarus colhidos no rio Chenab. A fragmentação do DNA em diferentes espécies de peixes de água doce é, portanto, um biomarcador confiável da poluição da água.

In vitro and in vivo genotoxicity assessment of ferric ferrocyanide and potassium-cobalt ferrocyanide.

Ferric hexacyanoferrate(II) (Fe4[Fe(CN)6]3), i.e. Prussian blue (PB) has been used for many years to remove from the body the two toxic isotopes of cesium and thallium following irradiation. Recently, potassium cobalt hexacyanoferrate(II) (K2COFe(CN)6), which has shown a better efficacy for decontamination, is also being considered for use to enhance the elimination of cesium isotopes. In view to its preclinical and clinical development, in vitro and in vivo GLP-compliant genotoxicity studies were carried out on this product as well as on PB for comparison. Several tests dissecting the main events leading to genotoxicity, i.e. mutagenicity and chromosomal aberrations, both structural and quantitative were implemented. In vitro, no mutagenic effect was observed in the Ames test but both compounds were positive in the mouse lymphoma assay on TK locus and induced clastogenic effects in the in vitro chromosomal aberrations test on human lymphocytes, either in absence or in presence of metabolic activation. K-Co-ferrocyanide was also assayed in vivo in the mouse bone marrow micronucleus assay and PB was assessed for DNA fragmentation in the rodent Comet assay in both glandular stomach and colon. In the in vivo micronucleus mouse bone marrow, K-Co-ferrocyanide did not display any genotoxic activity up to 2000 mg/kg/d (x2) by oral route. In opposite, PB induced a significant increase in DNA fragmentation both in the glandular stomach and in the colon of rat treated 3 times with intake ranging from 2000 to 500 mg/kg. PB should be considered as an in vivo mutagen as well as Potassium cobalt hexacyanoferrate(II) since the in vitro genotoxicity profiles of both ferrocyanides are quite similar. Their use as cesium/ thallium decontamination agents in human should be assessed following a benefit/risk approach to enable a robust decision-making.

Assessment of avian sperm DNA fragmentation using the sperm chromatin dispersion assay.

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen-thawed spermatozoa of each species were also incubated at 37°C for 5h and the within- and between-species variation of SDF, as an indicator of sperm DNA longevity, examined. The dynamic assessment of SDF using the SCDt revealed species and individual bird (rooster and turkey) differences in sperm DNA longevity.

DNA fragmentation of human spermatozoa: Simple assessment of single- and double-strand DNA breaks and their respective dynamic behavioral response.

BACKGROUND: Procedures to detect sperm DNA fragmentation (SDF), like the sperm chromatin dispersion (SCD) test, determine the "global" SDF without discriminating between spermatozoa with single-strand DNA breaks only (SDF-SSBs) and those containing double-strand DNA breaks (SDF-DSBs). OBJECTIVES: (a) To validate a test to distinguish human spermatozoa with massive DSBs (DSB-SCD assay), (b) to study the baseline SDF-SSBs and SDF-DSBs, and (c) to assess their dynamics in vitro. MATERIALS AND METHODS: (a) SDF-DSBs were determined by visualization of diffused DNA fragments from spermatozoa lysed under non-denaturing conditions. This was validated by in vitro incubation with DNase I and the comet assay. (b) Baseline SDF-DSBs and SDF-SSBs were determined in ejaculates from 95 males. (c) Their dynamic appearance was studied in samples untreated or exposed to hyperthermia, acidic pH, nitric oxide released by sodium nitroprusside (SNP), and the metabolic energy inhibitors 2-deoxy-D-glucose and antimycin A. RESULTS: (a) DNase I and comet assay experiments confirmed that the assay successfully determined SDF-DSBs. (b) The higher the SDF of the semen sample, the higher the frequency of SSBs, whereas DSBs behaved independently. Abnormal samples showed higher SDF than normozoospermic, the difference being only significant for SDF-SSBs. (c) During the first hours of incubation, the linear rate of increase in SDF-SSBs was 3.7 X higher than that of SDF-DSBs. All hazardous agents accelerated the SDF rate when compared to untreated spermatozoa, primarily being associated with SDF-SSBs. SNP treatment was the most damaging, rapidly inducing spermatozoa with SSBs which progressively evolved to DSBs. Remarkably, this phenomenon was also evidenced after acute SNP exposure, revealing cryptic sperm damage. CONCLUSION: The DSBs-SCD is an easy complement for SDF assessment. The dynamic study of SSBs and DSBs may improve the evaluation of sperm quality in clinical settings, particularly "unmasking" the presence of non-specific cryptic sperm damage that might otherwise go undetected.

Chitosan-mediated synthesis of biogenic silver nanoparticles (AgNPs), nanoparticle characterisation and in vitro assessment of anticancer activity in human hepatocellular carcinoma HepG2 cells.

The biopolymer chitosan is currently in widespread use because of its nontoxicity, biocompatibility and biodegradability. Therefore, in this study, chitosan extracted from shrimp shells was used to synthesise biogenic silver nanoparticles (AgNPs). UV-visible spectrophotometry of reduced silver nanoparticles in the colloidal solution showed a single peak at 400 nm, confirming the formation of AgNPs. The presence of biomolecules responsible for reducing and capping the biogenic AgNPs was confirmed by FTIR. Surface morphology of the biosynthesised AgNPs was characterised using SEM, and TEM analysis showed the formation of spherical shapes 17-50 nm. The presence of elemental silver in the synthesised biogenic AgNPs was confirmed using EDX and the crystalline structure characterised by XRD. Cytotoxicity of biogenic AgNPs was determined using MTT and the trypan blue exclusion assay. Morphological changes in HepG2 cells were detected by analysis of the DNA ladder pattern via gel electrophoresis, and the IC50 of HepG2 cell inhibition by AgNPs was 48 µg/ml. The upregulated caspase 3 and 9 protein expression results confirmed cell death via apoptosis. In conclusion, chitosan has the ability to synthesise AgNPs with in vitro apoptotic activities.

Assessment of heavy metals and its impact on DNA fragmentation in different fish species.

This study was conducted to assess water pollution by examining DNA fragmentation in selected fish organs (kidney, liver, gills, and muscle tissue) from Wallago attu, Sperata sarwari, Vulgaris vulgaris, and Labeo rohita collected from a known polluted section of the Chenab River, Pakistan, and from a control site. The fish were caught using a gill net and were assigned to three different weight groups (W1, W2, and W3) to study the degree of variation in DNA fragmentation in relation to body weight. In fish from the polluted site, DNA fragmentation was higher in kidney, liver, gills, and muscles, compared to the control. No significant DNA fragmentation was observed in fish collected from the control site. Highly significant (P < 0.01) relationship between body weight and DNA fragmentation was found in the organs of fish procured at the contaminated site. DNA fragmentation in body organs was found to be affected by the concentrations of lead, copper, nickel, and cadmium in W. attu, S. sarwari, L. rohita, and V. vulgarus harvested from Chenab River. DNA fragmentation in different freshwater fish species is therefore a reliable biomarker of water pollution.

Assessment of Neutrophil Apoptosis.

The process of neutrophil apoptosis has an important role in the resolution of acute inflammation. Apoptotic cell death is characterized by a coordinated sequence of cellular alterations that serve to uncouple neutrophil effector functions whilst maintaining plasma membrane integrity. In this way the release on neutrophil intracellular contents, including proteases, glycosidases, and reactive oxygen species, is limited during apoptosis. In addition, plasma membrane alterations associated with neutrophil apoptosis provide molecular cues that enable recognition by phagocytic cells, including macrophages. The recognition and uptake of apoptotic neutrophils by macrophages dampens proinflammatory responses to pathogen- or damage-associated molecular patterns and triggers release of proresolution mediators, that further promote resolution of inflammation. The key cellular and molecular events that act to control neutrophil apoptosis and subsequent macrophage phagocytosis have been characterized by in vitro studies, unveiling potential therapeutic targets for the manipulation of these regulatory pathways. In this chapter, we outline some of the key assays that are used to assess neutrophil apoptosis in vitro, together with methods to assess activation of the apoptotic machinery and phagocytic clearance of apoptotic neutrophils.

DNA fragmentation in concert with the simultaneous assessment of cell viability in a subfertile population: establishing thresholds of normality both before and after density gradient centrifugation.

PURPOSE: TUNEL assay is the most common, direct test for sperm chromatin integrity assessment. But, lack of standardized protocols makes interlaboratory comparisons impossible. Consequently, clinical thresholds to predict the chance of a clinical pregnancy also vary with the technique adopted. This prospective study was undertaken to assess the incidence of sperm DNA fragmentation in a subfertile population and to establish threshold values of normality as compared to a fertile cohort, both before and after density gradient centrifugation in the total and vital fractions. METHOD: Men presenting at a university hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. RESULTS: Total DNA fragmentation in the neat semen was significantly higher in the subfertile group, but the vital fraction was not significantly different between the two cohorts. After gradient centrifugation, DNA fragmentation increased significantly in the total fraction of the subfertile group but decreased significantly in the vital fraction. In the fertile cohort, there was a non-significant increase in total fragmentation and in the vital fraction the trend was unclear. CONCLUSIONS: Estimating total and vital sperm DNA fragmentation, after density gradient centrifugation, increased both the sensitivity and the specificity, thereby lowering the number of false negatives and false positives encountered. These findings provide opportunities to investigate the significance of the total and the vital fractions after different assisted reproductive technologies.

Bio-engineering and cellular imaging of silver nanoparticles as weaponry against multidrug resistant human pathogens.

'Go green' has also been implied to nanotechnology by harbouring eco-benign principle for a cleaner production of silver nanoparticles (AgNPs). This was achieved using a nitrate reducing Bacillus subtilis L1 (KT266579.1) inhabiting rhizosphere soil under optimized laboratory conditions, highlighting on its antibacterial modus operandi. Nano-characteristics and antimicrobial mechanism were investigated using spectroscopic and electron microscopic studies. Spectroscopic and microscopic characterization revealed typical surface plasmon resonance (SPR) with λmax 420 nm showing mean particle size of ~28.30 nm and spherical shaped nanoparticles. Antimicrobial susceptibility pattern of clinically important pathogens (n = 15) exposed to AgNPs at 10 µg, 15 µg and 20 µg/mL for 18 h was found significant in a dose dependent fashion. Electron and atomic force microscopic (AFM) studies have demonstrated the typical bactericidal effect of AgNPs (<25 µg/mL) associated with 'pitting effect', cell shrinkage and increase in surface roughness. The EDX spectrum of the control and treated bacteria showed the intrusion of AgNPs inside the bacterial cells endorsing the event of bacterial paralysis. DNA fragmentation assay demonstrated significant DNA damage in the form of smear, indicative of genotoxicity at ≤32 µg and ≤16 µg/mL of AgNPs respectively for Gram positive and negative strains in <12 h. These results suggest that AgNPs possess excellent antimicrobial activity, providing a potential lead for developing a broad spectrum antibacterial agent and extending its therapeutic modalities targeting antibiotic resistant strains at gene level.

Dynamic assessment of human sperm DNA damage II: the effect of sperm concentration adjustment during processing.

PURPOSE: To evaluate the effect of sperm concentration adjustment in human ejaculates on the sperm DNA quality and longevity. METHODS: Semen samples were obtained from 30 donors with a normal spermiogram. Following centrifugation, the sperm pellet was resuspended in PBS, and the sperm concentration adjusted to 200, 100, 50, 25, 12, and 6 × 106/mL. Each set of samples was incubated at 37 °C for 24 h, and the sperm DNA damage was assessed using the chromatin-dispersion test following 0 h, 2 h, 6 h, and 24 h of incubation. RESULTS: Sperm DNA fragmentation (SDF) did not differ between the selected experimental conditions at T0; however, Kaplan-Meier estimates for survival showed significant differences with respect to the dilution and time (all P values were smaller than .001). DNA fragmentation in semen samples adjusted to 200 × 106/mL was approximately 3.3 times higher when compared to samples containing 25 × 106/mL and 3.9 higher in comparison with samples adjusted to 12 × 106/mL following 2 h of in vitro incubation. Although there was evidence of individual variation in SDF during the incubation period, the general finding was that lower sperm concentrations resulted in a slower rate of DNA fragmentation. CONCLUSIONS: Incubation of spermatozoa for ART purposes should be done following a concentration adjustment below 25 × 106/mL in order to avoid a higher susceptibility of the sperm DNA molecule towards fragmentation.

Critical evaluation of two models of flow cytometers for the assessment of sperm DNA fragmentation: an appeal for performance verification.

Lack of standardized, reproducible protocols and reference values is among the challenges faced when using new or upgraded versions of instruments in reproductive laboratories and flow cytometry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay combined with flow cytometry routinely used for diagnostic measurement of sperm DNA fragmentation (SDF) is a unique example. Any change in the setting of the standard instrument, including upgrades of hardware or software, can lead to different results and may affect clinicians' decision for treatment. Therefore, we compared TUNEL results of SDF obtained from a standard (C6) flow cytometer with a newer version of the same instrument (C6 Plus) and examined the cutoff, sensitivity, and specificity without calibration (adjustment) and after adjustment. Identical sperm preparation and matched acquisition settings were used to examine the performance of two flow cytometers. The strength of agreement of the results between the two observers was also assessed. After adjustment of the settings, overall concordance became high and the two cytometers showed 100% positive and negative predictive value with 100% area under the curve. The overall correlation coefficient observed between C6 and C6 Plus was highly significant (P < 0.0001; r = 0.992; 95% confidence interval [CI]: 0.982-0.997). After adjustment, the two cytometers showed very high precision of 98% and accuracy of >99%. The interobserver agreement on C6 flow cytometer for the two observers was 0.801 ± 0.062 and 0.746 ± 0.044 for C6 Plus. We demonstrated a strong agreement between the samples tested on the two flow cytometers after calibration and established the robustness of both instruments.

A treatment algorithm for couples with unexplained infertility based on sperm chromatin assessment.

OBJECTIVE: To design a reproductive treatment algorithm based on the sperm DNA fragmentation (SDF) for couples with unexplained infertility following a poor intrauterine insemination (IUI) outcome. DESIGN: Couples that failed IUI with no apparent reproductive issue in both partners were allocated to diverse reproductive treatments on the basis of SDF. SETTING: Reproductive medical center in an academic setting. PATIENT(S): Over 4 years, couples with an unexpected poor IUI outcome and no apparent female or male partner reproductive issues were recruited. INTERVENTION(S): IUI, IVF, and ICSI were performed in the standard fashion following sperm SDF assays. MAIN OUTCOMES MEASURE(S): Fertilization rate, implantation rate, pregnancy characteristics, and delivery rates. RESULT(S): A total of 354 couples with unexplained infertility and normal semen parameters underwent 1133 IUI cycles. Clinical pregnancy rate (CPR) with IUI at our center in an age-matched cohort is 23.9% while the study cohort had 1.8%. Following SDF assessment, couples with failed IUI attempts but normal SDF (SCSA 9.8 ± 4.6%; TUNEL 11.8 ± 6.2%) underwent IVF with a CPR of 12.7%; those with abnormal SDF underwent ICSI with ejaculated spermatozoa, resulting in a CPR of 18.7%. This group included couples with normal SDF that had failed IVF. Couples with abnormal SDF that failed ICSI with ejaculated spermatozoa achieved a CPR of 31.0% with surgically retrieved spermatozoa. CONCLUSION(S): Couples with unexplained infertility that present with unexpectedly poor IUI outcomes can be funneled into a treatment algorithm guided by the integrity of the sperm genome for higher chances of pregnancy using an alternate method of insemination.

Safety assessment of sodium acetate, sodium diacetate and potassium sorbate food additives.

Cytotoxicity and genotoxicity of sodium acetate (SA), sodium diacetate (SDA), and potassium sorbate (PS) was tested on Human Umbilical Vein Endothelial Cells (HUVEC). Cytotoxicity was investigated by MTT assay and flow cytometry analysis, while genotoxicity was evaluated using DNA fragmentation and DAPI staining assays. The growth of treated HUVECs with various concentrations of SA, SDA and PS decreased in a dose-and time-dependent manner. The IC50 of 487.71, 485.82 and 659.96 µM after 24 h and IC50 of 232.05, 190.19 and 123.95 µM after 48 h of treatment were attained for SA, SDA and PS, respectively. Flow cytometry analysis showed that early and late apoptosis percentage in treated cells was not considerable. Also neither considerable DNA fragmentation nor DNA smear was observed using DAPI staining and DNA ladder assays. Overall, it can be concluded that the aforementioned food additives can be used as safe additives at low concentration in food industry.

In vivo assessment of the hepatotoxicity of a new Nostoc isolate from the Nile River: Nostoc sp. strain NRI.

Nostoc sp. is one of the most widely distributed cyanobacterial genera that produce potentially protein phosphatase (PP) inhibitor; microcystins (MCs). MCs have posed a worldwide concern due to predominant hepatotoxicity to human health. We have previously isolated a Nostoc strain (NR1) from the Nile River (the main water supply in Egypt) and this strain exerted production of rare and highly toxic MC; demethylated microcystin-LR. There is no data concerning risk factors of liver diseases for human and animal exposure to NR1-contaminated drinking water yet. It is thus important to evaluate acute (LD50 dose), subacute (0.01% and 10% of LD50 dose) and subchronic (0.01% and 10% of LD50 dose) hepatotoxicity's NR1 extract using experimental mice. Mice groups, who orally received 0.01% LD50, represented a permissible concentration of the World Health Organization (WHO) for MC in drinking water. Several parameters were detected, including hepatotoxicity (i.e. PP activity, liver function, oxidative stress markers and DNA fragmentation), pro-inflammatory cytokine (TNF-α) and liver histopathology. Our results demonstrated LD50 of NR1 extract was at 15,350 mg/kg body weight and caused hepatotoxicity that attributed to PP inhibition and a significant increase of hepatic damage biomarkers with lipid accumulation. Moreover, NR1 extract induced hepatic oxidative damage that may have led to DNA fragmentation and production of TNF-α. As demonstrated from the histopathological study, NR1 extract caused a severe collapse of cytoskeleton with subsequent focal degeneration of hepatocytes, necroinflammation and steatosis. The grade of hepatotoxicity in subacute (10% of LD50) group was higher than that in the subchronic (10% of LD50 and 0.01% of LD50, WHOch, respectively) groups. No significant hepatotoxicity was detectable for subacute (0.01% of LD50, WHOac) group. NR1 is therefore considered as one of the harmful and life-threatening cyanobacteria for Egyptian people being exposed to dose above WHO guideline. Thus, biological indicators and thresholds for water treatment are extremely needed.

Assessment of the cytotoxicity of ionic liquids on Spodoptera frugiperda 9 (Sf-9) cell lines via in vitro assays.

Cytotoxicity studies are important tools for the assessment of the toxicity of ionic liquids (ILs). In the present study, the cytotoxicity of eleven ILs against Spodoptera frugiperda 9 (Sf-9) cell lines were evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The effect on cellular morphology, ultrastructural morphology, and nuclear morphology induced by 1-ethyl-3-methylimidazolium bromide ([C2mim][Br]) was studied via inverted light microscopy observation, acridine orange staining, and transmission electron microscope (TEM) analysis, respectively. The effect on cell DNA fragmentation, cell apoptosis and cell cycle induced by [C2mim][Br] was also investigated via DNA agarose gel electrophoresis and flow cytometry analysis, respectively. The results showed that the cytotoxic effect of ILs on Sf-9 cells was related to the IL structures, concentrations, and length of exposure. The morphological features of apoptosis induced by [C2mim][Br] such as cell shrinkage and convolution, apoptotic bodies, pyknosis, and karyorrhesis were observed. All these phenomena confirmed that Sf-9 cells exposed to [C2mim][Br] died via apoptosis. This study complements the current knowledge about the cytotoxic properties of ILs on insect cells and highlights the mechanism by which ILs kill these cells. Furthermore, it provides a basis for further studies on the future applications of ILs as insecticides.

Assessment of hepatotoxicity of first-line anti-tuberculosis drugs on Wistar rats.

Adverse drug reactions are inevitable risk factors associated with use of modern medicines. First-line anti-tuberculosis drugs contribute to diverse pathological complications, and hepatotoxicity is one of them. This study investigated the effects of anti-TB drugs in combination (rifampicin [RIF] + isoniazid [INH] + pyrazinamide [PZA]) on Wistar rats. Rats were grouped as control group (saline), toxicant group that was given (30.85 mg/kg b.wt., INH + 61.7 mg/kg b.wt., RIF + 132.65 mg/kg b.wt. PZA in dosage extrapolated from dose that is used in human). Different anti-oxidant enzymes were measured in the liver along with histopathology, hematology, genotoxic effect on bone marrow chromosomes, and DNA fragmentation. In addition, gene and protein expression of CYP2E1, NR1I2, NAT, and CYP7A1 was measured by qPCR and western blot. After administration of anti-TB drugs to Wistar rats for 28 days, there was an increase in thiobarbituric acid reactive substances and a decrease in anti-oxidant enzymes. Marked changes in histopathology, hematology, DNA fragmentation, chromosomes, and in gene expression were observed. Results of the study proved increased hepatotoxicity due to combinational treatment of anti-TB drugs and also that CYP2E1, NR1I2, NAT, and CYP7A1 genes play a vital role in anti-TB drug-induced hepatotoxicity.