Isorhamnetin Influences the Viability, Superoxide Production and Interleukin-8 Biosynthesis of Human Colorectal Adenocarcinoma HT-29 Cells In Vitro.

Isorhamnetin has gained research interest for its anti-inflammatory, anti-proliferative and chemoprotective properties. In this study, human colon adenocarcinoma cells were cultured in the presence or absence of different isorhamnetin concentrations (5-150 µM) for 24 h or 48 h of cultivation to explore the impact on several parameters of viability/proliferation (mitochondrial function using an MTT test, metabolic activity, cell membrane integrity and lysosomal activity using a triple test). The intracellular generation of superoxide radicals using an NBT test and ELISA analysis was performed to observe the biosynthesis of interleukin 8 (IL-8) in cells stimulated with zymosan, as well as in basal conditions. The antiproliferative activity of isorhamnetin was demonstrated by significantly reduced values of mitochondrial and metabolic activity, integrity of cell membranes and lysosomal activity. Its high prooxidant potential was reflected by the significantly elevated generation of superoxides even in cells with low viability status. The anti-inflammatory effect of isorhamnetin was evident due to decreased IL-8 production, and the most significant decline in IL-8 concentration was observed after 24 h treatment in cells with induced inflammation. We demonstrated that isorhamnetin can suppress the proliferation of HT-29 cells, and this effect was correlated with pro-oxidative and anti-inflammatory activity of isorhamnetin.

Screening of Toxic Effects of Neonicotinoid Insecticides with a Focus on Acetamiprid: A Review.

Recently, neonicotinoids have become the fastest-growing class of insecticides in conventional crop protection, with extensive usage against a wide range of sucking and chewing pests. Neonicotinoids are widely used due to their high toxicity to invertebrates, simplicity, flexibility with which they may be applied, and lengthy persistence, and their systemic nature ensures that they spread to all sections of the target crop. However, these properties raise the risk of environmental contaminations and potential toxicity to non-target organisms. Acetamiprid is a new generation insecticide, which is a safer alternative for controlling insect pests because of its low toxicity to honeybees. Acetamiprid is intended to target nicotinic acetylcholine receptors in insects, but its widespread usage has resulted in negative impacts on non-target animals such as mammals. This review summarizes in vivo and in vitro animal studies that investigated the toxicity of specific neonicotinoids. With summarized data, it can be presumed that certain concentrations of neonicotinoids in the reproductive system cause oxidative stress in the testis; spermatogenesis disruption; spermatozoa degradation; interruptions to endocrine function and Sertoli and Leydig cell function. In the female reproductive system, acetamiprid evokes pathomorphological alterations in follicles, along with metabolic changes in the ovaries.

Comparison of the Effect of BPA and Related Bisphenols on Membrane Integrity, Mitochondrial Activity, and Steroidogenesis of H295R Cells In Vitro.

Bisphenol A (BPA) is an endocrine-disruptive chemical that is widely utilized in the production of polycarbonate plastic and epoxy resin, which are used to make a wide range of consumer products, food and drink containers, and medical equipment. When the potential risk of BPA emerged, it was substituted by allegedly less harmful substitutes such as bisphenols S, F, B, and AF. However, evidence suggests that all bisphenols can have endocrine-disruptive effects, while the extent of these effects is unknown. This study aimed to determine effect of BPA, BPAF, BPB, BPF, and BPS on viability and steroidogenesis in human adrenocortical carcinoma cell line in vitro. The cytotoxicity of bisphenols was shown to be considerable at higher doses. However, at low concentrations, it improved viability as well as steroid hormone secretion, indicating that bisphenols have a biphasic, hormetic effect in biological systems. The results are consistent with the hypothesis that bisphenols selectively inhibit some steroidogenic enzymes. These findings suggest that bisphenols have the potential to disrupt cellular steroidogenesis in humans, but substantially more detailed and systematic research is needed to gain a better understanding of the risks associated with bisphenols and their endocrine-disrupting effect on humans and wildlife.

Exceptional Properties of Lepidium sativum L. Extract and Its Impact on Cell Viability, Ros Production, Steroidogenesis, and Intracellular Communication in Mice Leydig Cells In Vitro.

The prevalence of reproductive dysfunction in males has risen in the last few years, and alternative therapies are gradually gaining in popularity. Our in vitro study aimed to evaluate the potential impact of Lepidium sativum L. on mice TM3 Leydig cells, concerning basal parameters such as cell viability, cell membrane integrity, and lysosomal activity, after 24 h and 48 h exposure. Moreover, reactive oxygens species generation, sex-steroid hormone secretion, and intercellular communication were quantified. In the present study, the microgreen extract from Lepidium was rich in ferulic acid, 4-OH benzoic acid, and resveratrol, with a significant antioxidant activity. The results showed that lower experimental doses (62.5-250 µg/mL) could positively affect the observed parameters, with significant differences at 250 µg/mL after 24 h and 48 h, respectively. Potential risks could be associated with higher concentrations, starting at 500 µg/mL, 1000 µg/mL, and 2000 µg/mL of Lepidium. Nevertheless, biochemical quantification indicated a significant antioxidant potential and a rich content of biologically active molecules at the applied doses, and time determined the intracellular response of the cultured model.

Comparative analysis of the detrimental in vitro effects of three fusariotoxins on the selected structural and functional characteristics of rabbit spermatozoa.

In this study, we evaluated the in vitro effects of 1-50 µM zearalenone (ZEA), deoxynivalenol (DON) and T-2 toxin (T-2) on rabbit spermatozoa for as much as 8 h of in vitro exposure. Our results indicate that all sperm quality parameters were negatively affected by these fusariotoxins in a time- and dose-dependent manner. The most prominent structure affected by ZEA was the plasma membrane, exhibiting alterations consistent with the onset of apoptosis and reactive oxygen species (ROS) overproduction. This correlated with the most prominent decline of the sperm motility among all selected fusariotoxins. Significant necrotic changes and mitochondrial dysfunction were primarily responsible for the sperm damage in the presence of T-2. Finally, exposure of spermatozoa to DON led to a significant decrease in the DNA integrity. This study may provide new information on the specific mechanisms of action involved in the in vitro toxic behavior of fusariotoxins on male gametes.

Inflammation, It's Regulation and Antiphlogistic Effect of the Cyanogenic Glycoside Amygdalin.

The inflammatory reaction accompanies in part or in full any disease process in the vascularized metazoan. This complicated reaction is controlled by regulatory mechanisms, some of which produce unpleasant symptomatic manifestations of inflammation. Therefore, there has been an effort to develop selective drugs aimed at removing pain, fever, or swelling. Gradually, however, serious adverse side effects of such inhibitors became apparent. Scientific research has therefore continued to explore new possibilities, including naturally available substances. Amygdalin is a cyanogenic glycoside present, e.g., in bitter almonds. This glycoside has already sparked many discussions among scientists, especially about its anticancer potential and related toxic cyanides. However, toxicity at different doses made it generally unacceptable. Although amygdalin given at the correct oral dose may not lead to poisoning, it has not yet been accurately quantified, as its action is often affected by different intestinal microbial consortia. Its pharmacological activities have been studied, but its effects on the body's inflammatory response are lacking. This review discusses the chemical structure, toxicity, and current knowledge of the molecular mechanism of amygdalin activity on immune functions, including the anti-inflammatory effect, but also discusses inflammation as such, its mediators with diverse functions, which are usually targeted by drugs.

Exogenous Factors Affecting the Functional Integrity of Male Reproduction.

Natural processes along with increased industrial production and the irresponsible behavior of mankind have resulted in environmental pollution. Environmental pollutants can be categorized based on their characteristics and appearance into the following groups: physical, biological, and chemical. Every single one of them represents a serious threat to the male reproductive tract despite the different modes of action. Male gonads and gametes are especially vulnerable to the effect of exogenous factors; therefore, they are considered a reliable indicator of environmental pollution. The impact of xenobiotics or radiation leads to an irreversible impairment of fertility displayed by histological changes, modulated androgen production, or compromised spermatozoa (or germ cells) quality. The present article reviews the exogenous threats, male reproductive system, the mode of action, and overall impact on the reproductive health of humans and animals.

The protective effect of stilbenes resveratrol and pterostilbene individually and combined with mycotoxin citrinin in human adenocarcinoma HT-29 cell line in vitro.

This study was focused to determine an individual and combined effect of mycotoxin citrinin (CIT) and two compounds of the stilbene family- resveratrol (RES) and his dimethyl ether analogue pterostilbene (PTE) which have many health benefits. As a model the human adenocarcinoma cell line HT-29 was used which may exhibits the properties of small intestine cells. Viability, plasma membrane integrity, lysosomal functionality, intracellular production of superoxide anions and superoxide dismutase activity were examined. The results indicate that concentrations of 50 and 100 µg/mL of the tested compounds were cytotoxic in mostly monitored parameters and probably caused apoptosis. HT-29 cells were more sensitive to PTE than to RES with a higher antioxidant effect of PTE than RES, which may be caused by its chemical structure. Both stilbenes at medium doses act as effective superoxide anions scavengers leading to reduction of oxidative stress and consequent cell damage. The nontoxic concentration of RES (25 µg/mL) protects the HT-29 cell line faced to the toxicity of CIT at 25 µg/mL by increasing viability of cells and by reducing the superoxide production induced by CIT concentrations of 12.5 µg/mL and 25 µg/mL.

Resveratrol attenuates hydrogen peroxide-induced oxidative stress in TM3 Leydig cells in vitro.

The objective of present study was to investigate in vitro protective potential of resveratrol in TM3 Leydig cells with induced oxidative stress using hydrogen peroxide (H2O2). Leydig cells experiencing oxidative stress exhibit reduced activities in androgens production, and become hypofunctional with age, which is also related to growing oxidative stress, while resveratrol has received growing attention as a cytoprotective agent. TM3 mouse Leydig cells were cultivated during 24 h in the presence of resveratrol (5, 10, 25, 50 and 100 µM) alone, or in combination with H2O2 (300/600 µM) to induce oxidative stress. Mitochondrial activity was evaluated using MTT test, triple assay was used in order to assess cell viability parameters, intracellular generation of superoxide was determined by the nitroblue-tetrazolium assay, and quantification of steroid hormones was performed by the enzyme- linked immunosorbent assay. Resveratrol alone treatment led to the most significantly improved values of all tested parameters in the cells of experimental group with addition of 10 µM of resveratrol in comparison to the control group. In the case of cells with induced oxidative stress (300 µM H2O2) resveratrol administration resulted in significantly increased (P < 0.05) metabolic activity, as well as cell membrane integrity at concentration 10 µM. Significantly improved (P < 0.001) lysosomal activity showed cells treated with 5 and 10 µM of resveratrol, and the level of both measured hormones was significantly higher (P < 0.05) in cells supplemented with 10 µM of resveratrol. Significant decline of superoxide radical production was observed in all experimental groups in comparison to the control exposed to H2O2 alone. With respect to cells exposed to higher concentration of H2O2 (600 µM), results showed positive effect of resveratrol only in biosynthesis of both androgens with significant increased values in experimental group treated with 5 µM (P < 0.05) and 10 µM (P < 0.01) of resveratrol, in addition, in the case of testosterone we recorded significant higher (P < 0.05) values in cells with addition of 25 and 50 µM resveratrol when compared to H2O2 control. More specific and systematic research focused especially on androgen biosynthesis is necessary related to the biological activity of resveratrol in male reproductive system due to inconsistent results of studies.

Identification of in vitro effect of 4-octylphenol on the basal and human chorionic gonadotropin (hCG) stimulated secretion of androgens and superoxide radicals in mouse Leydig cells.

The aim of our in vitro study was to assess the potential effect of 4-octylphenol (4-OP) on the basal and human chorionic gonadotropin (hCG)-stimulated cholesterol levels and biosynthesis of steroid hormones in cultured mouse Leydig cells. In addition, we evaluated the intracellular superoxide production following 4-OP treatment. Isolated mouse Leydig cells were cultured in the presence or absence of 1 IU/mL (hCG) with the addition of 0.04; 0.2; 1.0; 2.5 and 5.0 µg/mL 4-OP during 44 h. The level of cholesterol was determined from the culture medium using photometry. Quantification of steroid hormones was performed by the enzyme linked immunosorbent assay and intracellular generation of superoxide radicals was assessed by the nitroblue-tetrazolium assay. Administered concentrations of 4-OP (0.04-5.0 µg/mL) did not affect basal and hCG-stimulated cholesterol level significantly. However, basal DHEA secretion was increased significantly (P < 0.001) in the highest experimental dose (5 µg/mL) of 4-OP. By hCG-stimulated DHEA secretion, a significant (P < 0.001) decrease was recorded at 5.0 µg/mL 4-OP in comparison to the control group. The stimulatory effect of 4-OP was also confirmed in androstenedione secretion, when 2.5 and 5.0 µg/mL increased hormone secretion significantly (P˂0.05; P˂0.001). Exposure to experimental concentrations (0.04 to 5.0 µg/mL) of tested chemical reduced hCG-stimulated androstenedione formation, but not significantly. Measurements of basal testosterone production have shown significant (P˂0.01; P˂0.001) increase at 1.0; 2.5 and 5.0 µg/mL of 4-OP. Furthermore, 44 h treatment by 4-OP (1.0-5.0 µg/mL) caused significant (P˂0.01; P˂0.001) intracellular accumulation of superoxide radicals in exposed cells. A considerably more detailed and systematic research is required for a better understanding of risks associated with male reproductive system in humans and wildlife.

Parallel effect of 4-octylphenol and cyclic adenosine monophosphate (cAMP) alters steroidogenesis, cell viability and ROS production in mice Leydig cells.

Over the last decade, there is growing incidence of male reproductive malfunctions. It has been documented that numerous environmental contaminants, such as endocrine disruptors (EDs) may adversely affect the reproductive functions of humans as well as wildlife species. The aim of this in vitro study was to examine the effects of 4-octylphenol (4-OP) on the steroidogenesis in mice Leydig cells. We evaluated the impact of this endocrine disruptor on the cholesterol levels and hormone secretion in a primary culture. Subsequently, we determined the cell viability and generation of reactive oxygen species (ROS) following 4-OP treatment. Isolated mice Leydig cells were cultured in the presence of different 4-OP concentrations (0.04-5.0 µg/mL) and 1 mM cyclic adenosine-monophosphate during 44 h. Cholesterol levels were determined from the culture medium using photometry. Quantification of steroid secretion was performed by enzyme-linked immunosorbent assay. The cell viability was assessed using the metabolic activity assay, while ROS production was assessed by the chemiluminescence technique. Slightly increased cholesterol levels were recorded following exposure to the whole applied range of 4-OP, without significant changes (P>0.05). In contrast, the secretion of steroid hormones, specifically dehydroepiandrosterone, androstenedione, and testosterone was decreased following exposure to 4-OP. Experimental doses of 4-OP did not affect cell viability significantly; however a moderate decrease was recorded following the higher doses (2.5 and 5.0 µg/mL) of 4-OP. Furthermore, relative treatment of 4-OP (5.0 µg/mL) caused a significant (P < 0.001) ROS overproduction in the exposed cells.

Curcumin has protective and antioxidant properties on bull spermatozoa subjected to induced oxidative stress.

Over the past decades, there has been an emphasis on assessment of the use of natural compounds in the prevention or repair of oxidative injury to spermatozoa. Curcumin (CUR) is a natural phenol with powerful antioxidant properties. The aim of the present study was to examine if CUR could reverse reactive oxygen species (ROS)-mediated alterations to the motility, viability and intracellular antioxidant profile of bull spermatozoa subjected to a prooxidant (i.e., ferrous ascorbate - FeAA). Spermatozoa were washed from recently collected semen samples, suspended in 2.9% sodium citrate and subjected to CUR treatment (5, 10, 25 and 50µmol/L) in the presence or absence of FeAA (150µmol/L FeSO4 and 750µmol/L ascorbic acid) during a 6h in vitro culture. Spermatozoa motility characteristics were assessed using the SpermVision computer-aided spermatozoa analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified using luminometry and the nitroblue-tetrazolium (NBT) test was used to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the culture to assess the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). Treatment with FeAA led to a reduced spermatozoa motility (P<0.001), viability (P<0.001) and decreased the antioxidant characteristics of the samples (P<0.001) but increased the ROS generation (P<0.001), superoxide production (P<0.001) and lipid peroxidation (P<0.001). The CUR treatment led to a preservation of spermatozoa motion (P<0.001), mitochondrial activity (P<0.001) and antioxidant characteristics (P<0.05 with SOD and GSH; P<0.01 with CAT and GPx), revealing the concentration range of 25-50µmol/L CUR to be the most effective for sustaining spermatozoa viability. Data from the present study suggest that CUR exhibits significant protective and ROS-scavenging characteristics which may prevent oxidative insults to spermatozoa and thus preserve the functional activity of male gametes.