Nephrotoxicity evaluation of 3-monochloropropane-1,2-diol exposure in Sprague-Dawley rats using data-independent acquisition-based quantitative proteomics analysis.

3-Monochloropropane-1,2-diol (3-MCPD), as a heat-induced food process contaminant, possesses strongly toxic effect on kidney. The present study focuses on characterizing the proteome and clarifying the underlying molecular regulatory mechanisms in a model of kidney injury in rats treated with 3-MCPD. Data-independent acquisition (DIA)-mass spectrometry (MS) based proteomics was used to identify dysregulated proteins in kidney tissues of Sprague-Dawley (SD) rats treated with 30 mg/kg/day 3-MCPD by gavage for 28 days. It was found that a total of 975 proteins were deregulated after 3-MCPD treatment. Bioinformatic analyses revealed that several enzymes related to the metabolisms of amino acid, lipid and carbohydrate in endogenous metabolism were altered in response to 3-MCPD treatment. Moreover, some proteins involved in these pathways were also changed, mainly including oxidative stress, oxidative phosphorylation, apoptosis and autophagy. Our study unravels the vital roles of loss of mitochondrial homeostasis and function and cell death pathways in the development of renal damage induced by 3-MCPD, which provides further valuable insights into the initiation and resolution of 3-MCPD nephrotoxicity. The proposed DIA-MS workflow not only provides a choice for proteomic analysis in toxicological research, but also provides a more comprehensive understanding of the molecular mechanisms of nephrotoxicity induced by toxins.

Apoptosis is induced by sub-acute exposure to 3-MCPD and glycidol on Wistar Albino rat brain cells.

3-chloropropane-1,2-diol (3-MCPD) and its toxic metabolite glycidol were classified by the International Agency for Research on Cancer (IARC) as belonging to group 2B and 2A for humans. This study aimed to determine the sub-acute toxicity of these agents. Rats were exposed to 3-MCPD at 0.87 and 10 mg/kg/bw and glycidol (2,4 and 37,5 mg/kg/bw) for 90 days. miR-21 gene expression levels significantly decreased in all group's cerebellar tissues compared with control. Exposure to 10 mg/kg/bw 3-MCPD showed significant increases in PTEN in brain as compared to control group. The Akt gen expressions were significantly decreased in 3-MCPD and glycidol groups when compared to control group brains. Additionally, Caspase 3 and AIF immunopositivity significantly increased in 3-MCPD high dose and glycidol high dose groups in cerebellum granular layers compared to control. The results of the present study conclude that 3-MCPD and glycidol can induce apoptosis in rat brain tissue.

Necroptosis in 3-chloro-1, 2-propanediol (3-MCPD)-dipalmitate-induced acute kidney injury in vivo and its repression by miR-223-3p.

Fatty acid esters of 3-chloro-1, 2-propanediol (3-MCPD) are a group of processing-induced food contaminants with nephrotoxicity. This study investigated whether and how necroptosis played a role in the nephrotoxic effect of 3-MCPD-dipalmitate (2.5 g/kg BW) in C57 BL/6 mice. The results showed that the principal components in necroptosis pathway including receptor-interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) were up-regulated in 3-MCPD-dipalmitate-induced acute kidney injury (AKI). Deletion of RIPK3 or MLKL, and inhibition of RIPK1 suppressed AKI. The up-regulation of inflammatory cytokines in the kidney of 3-MCPD-dipalmitate treated mice were attenuated in RIPK3- or MLKL- deficient mice, suggesting a positive feedback loop involving necroptosis and inflammation. The microRNA analysis revealed that 38 known miRNAs and 40 novel miRNAs were differentially expressed (DE) in the kidney treated with 3-MCPD-dipalmitate. Of these miRNAs, miR-223-3p was significantly up-regulated during 3-MCPD-dipalmitate-induced AKI. In cultured mouse proximal tubular cells, a miR-223-3p mimic suppressed RIPK3 expression, which was blocked by miR-223-3p inhibitor. The luciferase reporter assay confirmed that miR-223-3p was able to inhibit RIPK3 expression by targeting the 3' un-translated region of RIPK3. These results suggest that necroptosis contributes to 3-MCPD-dipalmitate-induced acute kidney injury, and that may be attenuated by miR-223-3p.

DNA methylation analysis in rat kidney epithelial cells exposed to 3-MCPD and glycidol.

3-Monochloropropane-1,2-diol (3-MCPD) is a well-known food processing contaminant that has been regarded as a rat carcinogen, which is known to induce Leydig-cell and mammary gland tumors in males, as well as kidney tumors in both genders. 3-MCPD is highly suspected to be a non-genotoxic carcinogen. 2,3-Epoxy-1-propanol (glycidol) can be formed via dehalogenation from 3-MCPD. We aimed to investigate the cytotoxic effects of 3-MCPD and glycidol, then to demonstrate the possible epigenetic mechanisms with global and gene-specific DNA methylation in rat kidney epithelial cells (NRK-52E). IC50 value of 3-MCPD was determined as 48 mM and 41.39 mM, whereas IC50 value of glycidol was 1.67 mM and 1.13 mM by MTT and NRU test, respectively. Decreased global DNA methylation at the concentrations of 100 µM and 1000 µM for 3-MCPD and 100 µM and 500 µM for glycidol were observed after 48 h exposure by using 5-methylcytosine (5-mC) ELISA kit. Methylation changes were detected in promoter regions of c-myc and Rassf1a in 3-MCPD and glycidol treated NRK-52E cells by using methylation-specific PCR (MSP), whereas changes on gene expression of c-myc and Rassf1a were observed by using real-time PCR. However, e-cadherin, p16, VHL and p15 genes were unmethylated in their CpG promoter regions in response to treatment with 3-MCPD and glycidol. Alterations in DNA methylation might be key events in the toxicity of 3-MCPD and glycidol.

3-Monochloro-1,2-propanediol (3-MCPD) induces apoptosis via mitochondrial oxidative phosphorylation system impairment and the caspase cascade pathway.

3-Monochloro-1,2-propanediol (3-MCPD) is the most toxic chloropropanols compounds in foodstuff which mainly generated during thermal processing. Kidney is one of the primary target organs for 3-MCPD. Using human embryonic kidney cell (HEK293FT) as an in vitro model, we found that 3-MCPD caused concentration-dependent increase in cytoxicity as assessed by dye uptake, lactatedehydrogenase (LDH) leakage and MTT assays. HEK293FT cell treated with 3-MCPD suffered the decrease of mitochondrial membrane potential and the impairment of mitochondrial oxidative phosphorylation system, especially the reduced amount of mRNA expression and protein synthesis of electron transport chain complex II, complex IV, and complex III. More importantly, energy release (ATP synthesis) was significantly inhibited by 3-MCPD resulting from the down regulation expressions of ATP synthase (ATP6 and ATP8), as well as the loss of transmembrane potential required for synthesis of ATP. The decreased ratio of mitochondrial apoptogenic factors Bax/Bcl-2 and the cytochrome-c release from mitochondria to cytosol followed by the activation of apoptotic initiators caspase 9 and apoptotic executioners (caspase 3, caspase 6 and caspase 7) leading to apoptosis. The activation of caspase 8 and caspase 2 implied that there were probably other factors to induce the caspase-dependent apoptosis.

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Anopheles arabiensis egg treatment with dieldrin for sex separation leaves residues in male adult mosquitoes that can bioaccumulate in goldfish (Carassius auratus auratus).

The sterile insect technique (SIT) is a biological control tactic that is used as a component of area-wide integrated pest management (AW-IPM) programs. The SIT can only be applied against disease-transmitting mosquitoes when only sterile male mosquitoes are released, and the blood-sucking and potentially disease-transmitting females are eliminated from the production line. For Anopheles arabiensis, a potent vector of malaria, a genetic sexing strain was developed whereby females can be eliminated by treating the eggs or larvae with the insecticide dieldrin. To evaluate the presence of dieldrin residues in male mosquitoes designated for SIT releases, a simple, sensitive, and accurate gas chromatography-electron capture detector (GC-ECD) method was developed. In addition, bioaccumulation and food chain transfer of these residues to fish after feeding with treated mosquitoes was demonstrated. The overall recovery from method validation studies was 77.3 ± 2.2% (mean ± relative standard deviation [RSD]) for the mosquitoes, and 99.1 ± 4.4% (mean ± RSD) for the fish. The average dieldrin concentration found in adult male An. arabiensis was 28.1 ± 2.9 µg/kg (mean ± standard deviation [SD]). A range of 23.9 ± 1.1 µg/kg to 73.9 ± 5.2 µg/kg (mean ± SD) of dieldrin was found in the fish samples. These findings indicate the need to reassess the environmental and health implications of control operations with a SIT component against An. arabiensis that involves using persistent organochlorines in the sexing process.

Evaluation of the potential immunotoxicity of 3-monochloro-1,2-propanediol in Balb/c mice II. Effect on thymic subset, delayed-type hypersensitivity, mixed-lymphocyte reaction, and peritoneal macrophage activity.

3-Monochloro-1,2-propanediol (MCPD) is a well-known by-product of acid-hydrolyzed soy sauce during its manufacturing process. To evaluate the immunotoxicity of MCPD, we investigated its effect on the thymic subset, delayed-type hypersensitivity, mixed-lymphocyte reaction and peritoneal macrophage activity. MCPD was administered by gavage for 14 days at 0, 25, 50, and 100 mg/kg/day to female Balb/c mice. The thymic subsets and annexin-V positive cells in thymic cells were quantified by flow cytometry. Mixed-lymphocyte reaction, delayed-type hypersensitivity and peritoneal macrophage activity were assessed. The mixed-lymphocyte reaction and delayed-type hypersensitivity were not significantly changed. However, there were significant increases in the apoptosis of mice treated with high dose of MCPD compared to the vehicle control. A significant decrease in the CD4+CD8+ thymic subset of mice treated with high dose of MCPD was observed. The activity of peritoneal macrophage was significantly reduced in high dose group. These results indicate that MCPD could modulate the immune function in Balb/c mice.

Efficacy of lufenuron as chemosterilant against Ceratitis capitata in field trials.

Two field trials in citrus orchards in Turis (Valencia, Spain) and Denia (Alicante, Spain) were performed in order to test the sterilant effect of the insect growth regulator lufenuron against wild medfly Ceratitis capitata (Wiedemann) populations. Two application methods for lufenuron were tested: spraying, in spots, an emulsion of lufenuron in a protein bait, and hanging delta traps that contained a proteinaceous gel with lufenuron (solid bait). The sterilant effect was measured as medfly population reduction, reduction of fruit damage in treated fields, and the number of eggs hatching in punctured fruits. In order to assess the efficacy of lufenuron treatments, we recorded results obtained from two different zones in both trial fields: an outer zone, close to untreated fields, and an inner zone, in the centre of lufenuron treated fields. We observed a minimum sterilant effect in the outer zone and a maximum sterilant effect in the inner one. The maximum sterilant effect was in the inner zone, where a reduction of medfly population of 80.4% in the sprayed field and a reduction of 77.6% in the solid bait field was observed. In addition, the greater the distance from the untreated zones of the treated orchard (inwards), the lower the fruit damage and medfly population level. In this inner zone, fruit punctured by medfly developed significantly fewer larvae (38.8%) than punctured fruits from the outer zone (68.6%). In addition, we recorded the decline in the activity of the lufenuron treatments with time. Lufenuron activity persisted in field for at least 2 weeks with spray applications, and for 3 months with bait gels.

Antispermatogenic and antifertility effects of 20,25-diazacholesterol dihydrochloride in mice.

The effect of intraperitoneal administration for 28 days of 10, 20, and 30 mg/kg body weight per day of 20,25-diazacholesterol dihydrochloride (SC 12937), a hypocholesterolemic agent, on the testis of Parkes (P) strain mice was investigated. Histologically, testes in mice treated with 10 or 20 mg/kg body weight of SC 12937 showed non-uniform degenerative changes in the seminiferous tubules as both affected and normal tubules were observed in the same section; the affected tubules showed intraepithelial vacuolation, occurrence of giant cells, exfoliation of germ cells, and marginal condensation of chromatin in round spermatids. In both dosage groups, only 11-12% of the seminiferous tubules were affected, and no significant differences were found in the frequency of affected tubules between the two groups. By contrast, testes in mice treated with 30 mg/kg body weight of the drug exhibited a degenerated appearance of germ cells in all seminiferous tubules. The treatment also had adverse effects on motility, viability, morphology, and number of spermatozoa in the cauda epididymidis, and on fertility. Even 56 days after drug withdrawal, the above parameters remained markedly affected. Our results thus suggest that SC 12937 treatment causes antispermatogenic and antifertility effects in P mice and that the effects are not reversible up to 56 days after drug withdrawal. This compound may prove useful in the control of rodent populations.

Improved risk assessment by screening sperm parameters.

The question of whether a 4 or 9 week premating treatment period is more suitable in studies for effects on fertility and early embryonic development, and the extent to which the screening of sperm parameters may contribute to the detection of effects, has been under discussion since the ICH guideline changed in 1994/1995. This study presents a comparison between 4 and 9 weeks treatment with known male reproductive toxicants with regard to sperm motility, count, morphology, abnormal movements and testicular and epididymal histopathology. Mating outcome was examined after 4 weeks treatment. Three compounds with different targets and mechanisms of action were chosen: two testicular toxicants, Pyridoxine and Adriamycin and the epididymal toxicant, alpha-Chlorohydrine. Sperm motility was reduced in males treated with Pyridoxine (markedly) and alpha-Chlorohydrine (slightly) after 4 weeks treatment and in males treated with Adriamycin after 9 weeks treatment. With Pyridoxine and Adriamycin, sperm count was significantly increased after 4 weeks. Histopathological examination after 4 weeks showed characteristic changes leading to marked testicular tubular atrophy at 8/9 weeks, which was confirmed by a significantly reduced sperm count at 8/9 weeks. With alpha-Chlorohydrine, sperm count was not affected and the results of the histopathological examination were equivocal. Changes in sperm morphology were observed after 4/9 weeks of treatment with Pyridoxine. Mating outcome after 4 weeks was markedly affected with both Pyridoxine and alpha-Chlorohydrine, but no effect was observed with Adriamycin. The results of this study indicate that the two testicular toxicants would have been detected as male reproductive toxicants in a 4-week general toxicity study with routine testicular histopathology and examination of sperm parameters, without the need for mating trials. For the epididymal toxicant, alpha-Chlorohydrine, there was slightly reduced sperm motility after 4 weeks treatment, but mating trials were necessary for confirmation of the toxic effect. Without sperm motility examination, this effect would have been missed in early drug development causing problems in clinical studies. Further comparisons of the validity of 4 or 9 weeks treatment require the testing of other compounds with different targets/mechanism of actions, as well as evaluation of dose-response relationships.

Effect of glyzophrol on fertility of female black rats (Rattus rattus).

A mild degree of fibrosis, dilatation of blood vessels, haemorrhagic reactions in a few places, granulosa cells in the stroma and no effect on the maturation process of follicles were observed in the ovaries of 20 mg/kg of Glyzophrol treated rats. After 60 mg/kg of Glyzophrol treatment, the ovary revealed a reduction in the number of follicles, degenerative changes in existing follicles, frequent proliferation of granulosa cells and fibrosis in the stroma, along with karyorrhexis, karyolysis and pyknosis in the nuclei. Haemorrhage and dilated blood vessels in the stroma were well marked, suggesting that ovarian vasculature was affected by Glyzophrol. The ovary exhibited an increase in the activity of acid phosphatase after Glyzophrol treatment. Intense alkaline phosphatase activity was observed in the entire ovarian tissue after Glyzophrol treatment.

Effect of the chemosterilant bisazir on the eggs of Earias fabia stoll.

Bisazir-induced inhibition of nucleic acids and protein, and enhancement of free amino acids in the eggs of treated Earias fabia (Lepidoptera; Noctuidae) is reported. The degree of alterations in various crosses followed: T male X T female greater than UT male X T female greater than T male X UT female, which was also reflected in the weight loss of eggs. The alterations have been attributed to the abnormalities in the developing embryos due to genic imbalance in the spermatozoa, nurse cells and oocytes, ultimately leading to embryonic death.