Immunotoxicity of 2-Acetyl-4-tetrahydroxybutylimidazole in BALB/c mice with different vitamin B6 nutritional statuses.

Caramel color is a widely used food pigment, and 2-Acetyl-4-tetrahydroxybutylimidazole (THI) is a by-products of Class III caramel color. Some studies have shown that THI can reduce the number of peripheral blood lymphocytes. However, the comprehensive mechanism of THI immunotoxicity requires further study. In this study, the effects of THI on lymphocyte count, humoral immunity, cellular immunity and nonspecific immunity were determined and the effect of the nutritional status of VB6 on THI immunotoxicity was evaluated. Female BALB/c mice were divided into 3 groups and fed chow containing different doses of VB6: VB6-normal (6 mg/kg VB6), VB6-deprived (0.5 mg/kg VB6) or VB6-enhanced (12 mg/kg VB6) feed. Each group was further divided into 4 subgroups and treated with THI (0.5, 2.5 or 12.5 mg/kg bw) or the solvent control by gavage for 30 days. The thymic cortical thickness was measured with ViewPoint; the proportions of major immune cells and T cells in peripheral blood and tissues were detected via flow cytometry; the transformation and proliferation abilities of T and B cells were detected via T and B lymphocyte proliferation assays; NK cell activity was assessed via lactate dehydrogenase assays; humoral immune function was assessed via plaque-forming cell assays; and the immune function of T lymphocytes was assessed via delayed type hypersensitivity assays. The results showed that compared with those in the corresponding control group, the white blood cell count and lymphocyte count decreased significantly in all the VB6-deprived groups, in the 2.5 and 12.5 mg/kg VB6 groups, and in the 12.5 mg/kg VB6-enhanced group. With increasing THI dose, the thymic cortical layer became thinner. In the thymus, THI increased the proportions of CD3+ T cells and mature CD8+ T cells and decreased the proportions of immature double-positive, double-negative T cells and CD69-expressing lymphocytes. The proportions of naïve T cells and Tcm (central memory T) cells related to homing decreased. The proportion of mature T cells in the spleen decreased significantly. The proliferation of T cells stimulated by ConA decreased after THI exposure. VB6-deficient mice were more sensitive to THI immunotoxicity, and supplementation with VB6 had a certain protective effect on these mice. The results of the PFC and NK cell activity assays indicated that THI exposure might not affect humoral immune or innate immune function.

Chlorocholine chloride exposure induced spermatogenic dysfunction via iron overload caused by AhR/PERK axis-dependent ferritinophagy activation.

Chlorocholine chloride (CCC) is a plant growth regulator used worldwide that is detectable in cereals, fruits and animal products. The health effects of CCC exposure have raised public concern. Our previous research showed that CCC exposure decreased testosterone synthesis in pubertal rats. However, little is known about whether and how pubertal CCC exposure impacts spermatogenesis. In this study, we used BALB/c mice and spermatogonia-derived GC-1 cells to examine CCC-induced spermatogenic dysfunction. In vivo, pubertal CCC exposure led to decreased testicular weight, decreased testicular germ cells and poor sperm quality. This effect worsened after cessation of CCC exposure for the next 30 days. RNA-seq and western blot analysis revealed that CCC induced aryl hydrocarbon receptor (AhR) signaling, endoplasmic reticulum stress (ERS) and ferritinophagy. Increased iron content and lipid peroxidation levels were also observed in CCC-treated testes. In vitro, it was identified that iron overload mediated by enhanced ferritinophagy occurred in CCC-treated GC-1 cells, which might be attributed to the PERK pathway in ERS. Further, for the first time, our study elucidated the involvement of AhR in CCC-induced iron overload, which aggravated testicular oxidative damage via lipid peroxidation. Considering the adverse impact of CCC exposure on rodents, supportive evidence from GC-1 cells, and the critical importance of spermatogenesis on male development, the effects of CCC on the male reproduction warrant increased attention.

Chlormequat chloride induced activation of calmodulin mediated PI3K/AKT signaling pathway led to impaired sperm quality in pubertal mice.

Chlormequat chloride (CCC), as a widely used plant growth regulator, can cause impaired sperm quality and decreased testosterone synthesis in pubertal rats, but the underlying mechanism remains unclear. The purpose of this study was to elucidate the toxicokinetics and tissue distribution of CCC, as well as the possible mechanism of CCC-induced impairment in sperm quality. The concentration of CCC reached its peak 1 h after a single dose (200 mg/kg·bw) administration in mice plasma, and a bimodal phenomenon appeared in the testes, liver, and epididymis. In vivo, 200 mg/kg CCC caused testicular damage and impaired sperm quality in pubertal mice, and the expression of p-tyrosine and GSK3α decreased in cauda epididymidis, sperm and testes. CCC also caused the down-regulation of AKAP4 and the up-regulation of calmodulin (CaM), and activated the PI3K/AKT signaling pathway in the testes. In vitro, CCC reduced the levels of p-tyrosine, AKAP4 and GSK3α, increased the level of CaM and activated the PI3K/AKT signaling pathway in GC-1 cells. CaM antagonist (W-7 hydrochloride) and PI3K inhibitor (LY294002) can effectively improve the expression of GSK3α and AKAP4 by suppressing the PI3K/AKT signaling pathway in GC-1 cells treated with CCC. It was indicated that CCC induced impairment in sperm quality might be partially related to the activation of PI3K/AKT signaling pathway mediated by CaM.

Effect of lanthanum nitrate on adipogenesis in mice.

Lanthanum (La) is widely used in modern industry and agriculture because of its unique physicochemical properties and is broadly exposed in the population. Some studies have shown that La may have some effects on adipogenesis, but there is a lack of related in vivo evidence. In this study, the effects of La(NO3 )3 on adipogenesis and its associated mechanism were studied using C57BL/6J mouse model. The results showed that La(NO3 )3 exposure caused a decrease in body weight and the percentage of fat content in mice. In addition, the adipose marker molecules and specific adipogenic transcription factors decreased in both white adipose tissue (WAT) and brown adipose tissue (BAT). Detection of signaling pathway-related molecules revealed that canonical wnt/ß-catenin pathway-related molecules were upregulated in both adipose tissues. In summary, in vivo exposure to La(NO3 )3 might inhibited adipogenesis in mice, possibly through upregulation of the canonical Wnt/ß-catenin signaling pathway.

Effects of 2-acetyl-4-tetrahydroxybutylimidazole exposure during gestation and lactation on maternity and offspring immune function in Balb/c mice.

2-Acetyl-4-tetrahydroxybutylimidazole (THI), a by-product of Class Ⅲ caramel color, is generally recognized to cause lymphopenia in mammals. However, it remains unknown whether THI exposure during gestation and lactation causes damage to the immune system of offspring. In this study, pregnant Balb/c mice were gavaged with 0, 0.5, 2.5 and 12.5 mg/kg THI from gestation day (GD) 6 to postanal day (PND) 21, after which we treated another batch of dams from GD6 to PND21 and the offspring for 3 weeks after weaning with 0, 2, 10, 50 mg/L THI in drinking water respectively, and investigated the immunological anomalies of dams and offspring. The results showed that lymphopenia was observed in dams but not in weaning pups on PND21, which were exposed to THI during gestation and lactation. 2 mg/L THI and 2.5 mg/kg THI began to cause a remarkable reduction of the numbers of white blood cells and lymphocytes in dams. Besides both the cellular and the humoral immune response was not affected in weaning pups, which were measured by plaque-forming cell (PFC) assay and delayed-type hypersensitivity (DTH) assay respectively. Furthermore, THI could be detected in the plasma of dams with a dose-dependent manner, but not in that of both female and male weaning pups. In both male and female offspring being treated with 10 and 50 mg/L THI for another 3 weeks after weaning, lymphocytopenia was observed and T lymphocytes including CD4+ and CD8+ cells were significantly reduced in their spleens except lymph nodes. 10 and 50 mg/L THI treatment increased CD4+ and CD8+ single positive cells in thymus of female and male weaning mice. Mitogen-induced proliferation ability of T cells in the spleen and lymph nodes was impaired in female weaning mice exposed 50 mg/L THI, while male weaning mice treated with 10 and 50 mg/L THI showed impairment in the spleen but not lymph nodes. Based on the results in this study, no observed adverse effect level (NOAEL) for 3-week THI treatment in weaning mice was considered to be 2 mg/L (0.30 mg/kg bw for female mice and 0.34 mg/kg bw for male mice). And NOAEL for THI treatment in dams might be set to 0.5 mg/kg bw/day. Collectively from the perspective of NOAEL, offspring are not more sensitive than dams or adult mice.

The unfixed light pattern contributes to depressive-like behaviors in male mice.

Light pollution is now associated with an increased incidence of mental disorders in humans, and the unfixed light pattern (ULP) is a common light pollution that occurs in such as rotating shift work. However, how much contribution the ULP has to depression and its potential mechanism are yet unknown. Our study aimed to investigate the effect of the ULP on depressive-like behaviors in mice and to explore the links to the circadian-orexinergic system. Male C57BL/6 J mice were exposed to the ULP by subjecting them to an alternating light pattern every 6 days for 54 days. The tail suspension test (TST) and forced swimming test (FST) were conducted to assess depressive-like behaviors. The rhythm of locomotor activity and the circadian expression of cFOS in the suprachiasmatic nucleus (SCN), clock genes in the liver, and corticosterone (CORT) in serum were detected to observe changes in the circadian system. The circadian expression of orexin-A (OX-A) in the lateral hypothalamus area (LHA) and dorsal raphe nucleus (DRN) and serotonin (5-HT) in the DRN were measured to determine alterations in the orexinergic system. The results showed that mice exposed to the ULP exhibited increased immobility time in the TST and FST. The ULP significantly disrupted the circadian rhythm of locomotor activity, clock genes in the liver, and CORT in the serum. Importantly, when exposed to the ULP, cFOS expression in the SCN showed decreased amplitude. Its projection area, the LHA, had a lower mesor of OX-A expression. OX-A projection to the DRN and 5-HT expression in the DRN were reduced in mesor. Our research suggests that the ULP contributes to depressive-like behaviors in mice, which might be related to the reduced amplitude of circadian oscillation in the SCN and hypoactivity of the orexinergic system. These findings may provide novel insights into rotating shift work-related depression.

Lanthanum nitrate inhibits adipogenesis in 3T3-L1 preadipocytes with a disorder of mitotic clonal expansion process.

Lanthanum (La) as a rare earth element is widely used in agriculture, industry, and medicine. It has been suggested in several studies that La might influence glycolipid metabolism in vivo. In this study, we used 3T3-L1 preadipocytes as in vitro cell model to elucidate the effects of La(NO3 )3 on adipogenesis and the underlying mechanisms. The results showed that La(NO3 )3 could inhibit the adipogenic differentiation of 3T3-L1 preadipocytes, which showed a decrease in lipid accumulation and the downregulation of specific adipogenic transcription factors. La(NO3 )3 exerted its inhibitory effect mainly at the early differentiation stage. Furthermore, La(NO3 )3 influenced the S-phase entry and cell cycle process during the mitotic clonal expansion and regulated the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and expressions of the proteins in phosphatidylinositol 3-kinase (PI3K)/Akt pathway at the early stage of differentiation. Besides, La(NO3 )3 upregulated the expressions of wnt10b mRNA and ß-catenin protein and promoted the nucleus translocation of ß-catenin. Additionally, we found that La(NO3 )3 could promote the growth of 3T3-L1 preadipocytes both with and without MDI (3-isobutyl-1-methylxanthine [IBMX], dexamethasone [Dex], and insulin) stimulation. Collectively, these results indicated that La(NO3 )3 could inhibit adipogenesis in 3T3-L1 preadipocytes and influence cell proliferation.

Chlorocholine chloride induced testosterone secretion inhibition mediated by endoplasmic reticulum stress in primary rat Leydig cells.

Chlorocholine chloride (CCC) is well acknowledged as a plant growth regulator and may be considered as a potential environmental endocrine disrupting chemical. In our previous studies, it was found that CCC exposure at a pubertal stage reduced the serum and testicular levels of testosterone, decreased the sperm motility and delayed the puberty onset. However, the molecular mechanisms of CCC-induced testosterone secretion disorders remain unclear. In this study, we found that CCC exposure above 20 µg/mL inhibited the secretion of testosterone in Sprague-Dawley rats Leydig cells. Proteomic and pathway enrichment analysis indicated that CCC might induce endoplasmic reticulum (ER) stress. Western blot detection showed CCC exposure at 100, 200 µg/mL increased the protein level of glucose-regulated protein 78 (GPR78), C/EBP-homologous protein (CHOP), the ubiquitin-conjugating enzyme E2 D1 (UBE2D1) and the ring finger protein (RNF185) in the Leydig cells. The Leydig cells treated with 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, rescued the testosterone secretion disorders and alleviated CCC-induced increase in the ER stress related protein levels at 200 µg/mL CCC treatment. Overall, CCC in vitro exposure might disturb testosterone production of Leydig cells and endoplasmic reticulum stress was involved in it.

Effects of 4-nonylphenol on adipogenesis in 3T3-L1 preadipocytes and C3H/10T1/2 mesenchymal stem cells.

Obesogens are a subset of endocrine disruptor chemicals (EDCs) that cause obesity. The typical EDC 4-nonylphenol (4-NP) has been identified as an obesogen. However, the in vitro effects of 4-NP on adipogenesis remain unclear. In this study, 3T3-L1 preadipocytes and C3H/10T1/2 mesenchymal stem cells (MSCs) were used to investigate the influence of 4-NP on adipogenesis. The differentiation protocols for 3T3-L1 preadipocytes and C3H/10T1/2 MSCs took 8 and 12 days, respectively, beginning at Day 0. In differentiated 3T3-L1 preadipocytes, 20 µM 4-NP decreased cell viability on Days 4 and 8. Exposure to 4-NP inhibited triglyceride (TG) accumulation and adipogenic marker expression on Days 0-8, but the inhibitory effects were weaker on Days 2-8. The protein expression of pSTAT3 or STAT3 decreased on Days 0-8 and 2-8. Conversely, 4-NP promoted TG accumulation and the adipogenic marker expression in C3H/10T1/2 adipocytes. The opposing effects were attributed to physiological differences between the two cell lines. The 3T3-L1 preadipocytes are dependent on mitotic clonal expansion (MCE) to drive differentiation, while C3H/10T1/2MSCs and human preadipocytes are not. Additionally, 4-NP downregulated ß-catenin expression in C3H/10T1/2 adipocytes. Accordingly, we hypothesized that 4-NP promotes adipogenesis. The role of the canonical Wnt pathway in the promotion of adipogenesis by 4-NP requires further validation. This study provides new insights into the mechanisms and appropriate risk management of 4-NP.

Effects of prenatal chlorocholine chloride exposure on pubertal development and reproduction of male offspring in rats.

Chlorocholine chloride (CCC) promote plant growth as a regulator. Emerging evidence by our group showed that CCC might restrain the puberty onset and impair the reproductive functions in male rats through HPT axis. In this study, we further investigated the effects of prenatal CCC exposure on pubertal development, reproduction of male offspring in rats and explored the underlying mechanisms. The results showed that CCC of 137.5 and 200 mg/kg bw/day delayed the age of preputial separation (PPS), decreased the sperm motility of male offspring. PP1γ2 which is an essential protein in spermatogenesis reduced in 137.5 and 200 mg/kg bw/day groups. Crucial hormones involved in hypothalamic-puititary-testicular (HPT) axis decreased at postnatal day (PND) 30. It was indicated that CCC exposure in pregnancy might disturb the pubertal development, reproductive functions of male offspring through HPT axis and disturb the sperm motility through PP1γ2.

Maternal chlormequat chloride exposure disrupts embryonic growth and produces postnatal adverse effects.

We showed previously that chlormequat chloride, a widely used plant growth regulator, could affect embryonic growth and growth hormone (GH)-insulin-like growth factor 1 (IGF-1) axis of rats. However, the potential effects of low dose chlormequat chloride exposure during pregnancy on embryonic and postnatal growth and development remain unclear. To further assess the risk of chlormequat chloride to human embryonic growth and postnatal health, we exposed maternal rats orally to the chemical during pregnancy at 5 mg/kg bw, a dose corresponding to the human acceptable daily intake (ADI) level set by World Health Organization (WHO), and determined the effects of chlormequat on embryo growth and postnatal health. We found that chlormequat chloride increased embryonic growth parameters, GH, and GH-releasing hormone (GHRH) levels, but did not affect somatostatin and IGF-1 on gestational day (GD) 11. In the pups of postnatal day (PD) 7, we observed increased head length, decreased body fat percentage, hypoglycemia, hyperlipidemia and hyperproteinemia. In conclusion, maternal exposure to chlormequat chloride during pregnancy disrupts the embryonic growth probably through its effects on growth regulators and even has adverse effects on postnatal health.

Blue Light Deprivation Produces Depression-Like Responses in Mongolian Gerbils.

BACKGROUND: Depression is a leading cause of disability worldwide and is a major contributor to the overall global burden of disease, but its etiology is poorly understood. It has been reported that a disrupted biological rhythm, in terms of a shortened light duration and total darkness, can cause depression-like behaviors in animals. Blue light was reported to have an inhibitory effect on melatonin, which is considered an important clock rhythm biomarker. In the present study, we investigated the effects of blue light deprivation on depressive-like behaviors in gerbils and explored the underlying mechanisms. METHODS: Gerbils were housed under white light with a filter to block the blue light or without a filter. The behaviors of the gerbils were observed. The biological rhythm, 5-HT, hypothalamic-pituitary-adrenal (HPA) axis and melanopsin pathway were analyzed. RESULTS: We found that blue light deprivation (BLD) induced depression-like behavior in gerbils. Melatonin lost its rhythm, and corticosterone (CORT) levels decreased in the morning in the BLD group. Lower corticotropin-releasing hormone (CRH) in the hypothalamus and lower adrenocorticotropin hormone (ACTH)/CORT in serum were observed after BLD. Furthermore, 5-HT in the serum and brain were decreased after BLD. Additionally, BLD affected the blue light sensitivity protein melanopsin and its pathway, with downregulation of the proteins melanopsin, PKCα, and c-Fos and the mRNA levels of c-fos and trpc3 and upregulation of the protein p-PKCα. CONCLUSIONS: Our findings indicated that BLD might produce depression-like behaviors in gerbils. Melatonin arrhythmicity, HPA axis abnormalities, 5-HT decreases and melanopsin pathway changes might be associated with the depression behavioral phenotype in gerbils.

Effects of chlorocholine chloride on pubertal development and reproductive functions in male rats.

Chlorocholine chloride (CCC), a plant growth retardant, may act as an endocrine disruptor. Our previous study showed that pubertal CCC exposure in rats might decrease testosterone (T) synthesis. This study observed the changes in pubertal development and reproduction of male rats exposed to CCC and its underlying mechanisms. Rats were exposed to CCC (0, 75, 137.5 and 200 mg/kg bw/day) from postnatal day 23 to 60. The results showed that CCC treatment delayed the onset of puberty and reduced the relative organ weight of prostate. Seminiferous tubules with deciduous spermatogenic cells were observed in the 200 mg/kg bw/day group. Sexual behavior was inhibited in the 137.5 and 200 mg/kg bw/day groups. Sperm motility, litter size and normalized anogenital distance (AGD) of male pups were decreased in the 137.5 and 200 mg/kg bw/day groups. Serum kisspeptin level and serum and testicular levels of T were reduced in all CCC treated groups. Crucial hormones in hypothalamic-pituitary-testicular (HPT) axis were reduced subsequently after CCC treatment. Collectively, our results demonstrated that CCC might disturb HPT axis through suppressing the secretion of kisspeptin and subsequently lead to delayed puberty onset and impaired reproductive functions.

Chlormequat chloride promotes rat embryonic growth and GH-IGF-1 axis.

Chlormequat chloride, a plant growth regulator, is widely applied in agriculture because it can promote sturdier growth of the crops. In this research, we found that rat embryo growth on GD11 was inhibited in vitro at 50 µg/ml but promoted in vivo at 75 mg/kg.bw by maternal oral exposure. Therefore, the concentrations of chlormequat chloride in the sera of the pregnant rats on gestation day (GD)11 were determined by a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) test to be 1.94 ± 0.023 µg/ml, 3.84 ± 0.080 µg/ml, and 7.08 ± 0.11 µg/ml, respectively, when the pregnant rats were orally exposed to chlormequat chloride at 75, 137.5, and 200 mg/kg.bw. Hence, we performed WEC tests again and confirmed that the rat embryo growth in vitro was promoted by chlormequat chloride at 5 µg/mL. The embryonic growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels were increased by chlormequat chloride both in vitro and in vivo compared with the control ones. We concluded that chlormequat chloride could elevate GH and IGF-1 levels in embryos and promote embryonic growth both in vitro and in vivo.