Maternal exposure to 4-vinylcyclohexene diepoxide during pregnancy leads to disorder of gut microbiota and bile acid metabolism in offspring.

Our previous study reveals that maternal exposure to 4-vinylcyclohexene diepoxide (VCD) during pregnancy causes insufficient ovarian follicle reserve and decreased fertility in offspring. The present study aims to further explore the reasons for the significant decline of fecundity in mice caused by VCD, and to clarify the changes of gut microbiota and microbial metabolites in F1 mice. The ovarian metabolomics, gut microbiota and microbial metabolites were analyzed. The results of ovarian metabolomics analysis showed that maternal VCD exposure during pregnancy significantly reduced the concentration of carnitine in the ovaries of F1 mice, while supplementation with carnitine (isovalerylcarnitine and valerylcarnitine) significantly increased the number of ovulation. The results of 16 S rDNA-seq and microbial metabolites analysis showed that maternal VCD exposure during pregnancy caused disordered gut microbiota, increased abundance of Parabacteroides and Flexispira bacteria that are involved in secondary bile acid synthesis. The concentrations of NorDCA, LCA-3S, DCA and other secondary bile acids increased significantly. Our results indicate that maternal exposure to VCD during pregnancy leads to disorder in gut microbiota and bile acid metabolism in F1 mice, accompanying with decreased ovarian function, providing further evidence that maternal exposure to VCD during pregnancy has intergenerational deleterious effects on offspring.

APOE4 genotype exacerbates the impact of menopause on cognition and synaptic plasticity in APOE-TR mice.

The impact of sex and menopausal status in Alzheimer's disease remains understudied despite increasing evidence of greater female risk, particularly in APOE4 carriers. Utilizing female APOE-TR mice maintained on a high-fat diet background we induced ovarian failure through repeated VCD injections, to mimic human menopause. At 12 months of age, recognition memory and spatial memory were assessed using object recognition, Y-maze spontaneous alternation, and Barnes maze. A VCD*genotype interaction reduced the recognition memory (P < .05), with APOE4 VCD-treated animals unable to distinguish between novel and familiar objects. APOE4 mice displayed an additional 37% and 12% reduction in Barnes (P < .01) and Y-maze (P < .01) performance, indicative of genotype-specific spatial memory impairment. Molecular analysis indicated both VCD and genotype-related deficits in synaptic plasticity with BDNF, Akt, mTOR, and ERK signaling compromised. Subsequent reductions in the transcription factors Creb1 and Atf4 were also evident. Furthermore, the VCD*genotype interaction specifically diminished Ephb2 expression, while Fos, and Cnr1 expression reduced as a consequence of APOE4 genotype. Brain DHA levels were 13% lower in VCD-treated animals independent of genotype. Consistent with this, we detected alterations in the expression of the DHA transporters Acsl6 and Fatp4. Our results indicate that the combination of ovarian failure and APOE4 leads to an exacerbation of cognitive and neurological deficits.

Effects of low intensity pulsed ultrasound on expression of B-cell lymphoma-2 and BCL2-Associated X in premature ovarian failure mice induced by 4-vinylcyclohexene diepoxide.

BACKGROUND: Premature ovarian failure (POF) is a common disease in the field of Gynecology. Low intensity pulsed ultrasound (LIPUS) can promote tissue repair and improve function. This study was performed to determine the effects of LIPUS on granulosa cells (GCs) apoptosis and protein expression of B-cell lymphoma-2 (Bcl-2) and BCL2-Associated X (Bax) in 4-vinylcyclohexene diepoxide (VCD)-induced POF mice and investigate the mechanisms of LIPUS on ovarian function and reserve capacity. METHODS: The current POF mice model was administrated with VCD (160 mg/kg) by intraperitoneal injection for 15 consecutive days. The mice were divided into the POF group, LIPUS group and control group. In the LIPUS group, the right ovary of mice was treated by LIPUS (acoustic intensity was 200 mW/cm2, frequency was 0.3 MHz, and duty cycle was 20%) for 20 min, 15 consecutive days from day 16. The mice of the POF group and control group were treated without ultrasonic output. The basic observation and body weight were recorded. Hematoxylin and eosin staining (H&E staining) and enzyme-linked immunosorbent assay (ELISA) were applied to detect ovarian follicle development, ovarian morphology and sex hormone secretion. Ovarian GCs apoptosis was detected by TUNEL assay and immunohistochemistry. RESULTS: The results showed that VCD can induce estrus cycle disorder, follicular atresia, sex hormone secretion decreased and GCs apoptosis in mice to establish POF model successfully. LIPUS significantly promoted follicular development, increased sex hormone secretion, inhibited excessive follicular atresia and GCs apoptosis. The mechanism might be achieved by increasing the protein expression of Bcl-2 and decreasing the expression of Bax in ovaries. CONCLUSIONS: LIPUS can improve the POF induced by VCD. These findings have the potential to provide novel methodological foundation for the future research, which help treat POF patients in the clinic.

Linalool- and α-terpineol-induced programmed cell death in Chlamydomonas reinhardtii.

Plant allelochemicals effectively inhibit and/ or control algal growth, and have potential to use as algaecide. To uncover the lethal mechanism of 2 anti-algal compounds linalool and α-terpineol identified from Cinnamomum camphora extracts, and promote their development as algaecide, the H2O2 production, photosynthetic abilities, caspase-like activities, nuclear changes and DNA degradation were investigated in Chlamydomonas reinhardtii treated with the 2 compounds. H2O2 content burst in linalool treatment at 0.5 h and in α-terpineol treatment at 1 h, with increases of 2.7 folds and 1.3 folds, respectively, compared to that at 0 h. The photosynthetic pigments gradually degraded, and Fv/Fm gradually declined to zero, indicating that the cell death was not a necrosis due to the gradual disappearance of physiological process. In C. reinhardtii cells, the caspase-9-like and caspase-3-like were activated in the treatments with the 2 compounds for 1 h. With prolonging the treatment time, the fluorescent intensity of the cell nucleuses stained by DAPI gradually enhanced and then faded, and the genomic DNA isolated from the cells gradually degraded. These hallmarks indicated that the death of C. reinhardtii cells in linalool and α-terpineol treatments was a programmed cell death (PCD) triggered by the increased reactive oxygen species (ROS). Compared to α-terpineol treatment, linalool treatment showed stronger promoting effects on PCD at the same time point, which may be caused by the higher ROS content inducing higher caspase-9-like and caspase-3-like activities in a short time.

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models.

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3'-methoxyavarone, 4'-(methylamino)avarone and 3'-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3'-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3'-methoxyavarone and 3'-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.

Redistribution of NMDA Receptors in Estrogen-Receptor-ß-Containing Paraventricular Hypothalamic Neurons following Slow-Pressor Angiotensin II Hypertension in Female Mice with Accelerated Ovarian Failure.

Hypertension in male and aging female rodents is associated with glutamate-dependent plasticity in the hypothalamus, but existing models have failed to capture distinct transitional menopausal phases that could have a significant impact on the synaptic plasticity and emergent hypertension. In rodents, accelerated ovarian failure (AOF) induced by systemic injection of 4-vinylcyclohexane diepoxide mimics the estrogen fluctuations seen in human menopause including the perimenopause transition (peri-AOF) and postmenopause (post-AOF). Thus, we used the mouse AOF model to determine the impact of slow-pressor angiotensin II (AngII) administration on blood pressure and on the subcellular distribution of obligatory N-methyl-D-aspartate (NMDA) receptor GluN1 subunits in the paraventricular hypothalamic nucleus (PVN), a key estrogen-responsive cardiovascular regulatory area. Estrogen-sensitive neuronal profiles were identified in mice expressing enhanced green fluorescent protein under the promoter for estrogen receptor (ER) ß, a major ER in the PVN. Slow-pressor AngII increased arterial blood pressure in mice at peri- and post-AOF time points. In control oil-injected (nonhypertensive) mice, AngII decreased the total number of GluN1 in ERß-containing PVN dendrites. In contrast, AngII resulted in a reapportionment of GluN1 from the cytoplasm to the plasma membrane of ERß-containing PVN dendrites in peri-AOF mice. Moreover, in post-AOF mice, AngII increased total GluN1, dendritic size and radical production in ERß-containing neurons. These results indicate that unique patterns of hypothalamic glutamate receptor plasticity and dendritic structure accompany the elevated blood pressure in peri- and post-AOF time points. Our findings suggest the possibility that distinct neurobiological processes are associated with the increased blood pressure during perimenopausal and postmenopausal periods.

Toxicity of Monoterpene Structure, Diversity and Concentration to Mountain Pine Beetles, Dendroctonus ponderosae: Beetle Traits Matter More.

A high diversity of plant defenses may be a response to herbivore diversity or may be collectively more toxic than single compounds, either of which may be important for understanding insect-plant associations. Monoterpenes in conifers are particularly diverse. We tested the fumigant toxicity of four monoterpenes, alone and in combination, to mountain pine beetles, Dendroctonus ponderosae, in the context of the beetles' individual body traits. Chemical structures of tested monoterpene hydrocarbons had modest effects on beetle survival, mass loss, water content and fat content, with (R)-(+)-limonene tending to be more toxic than (-)-α-pinene, (-)-ß-pinene, and (+)-3-carene. Monoterpene diversity (all qualitative combinations of one to four monoterpenes) did not affect toxicity. Concentration (0 to 1200 ppm) of individual monoterpenes was a strong determinant of toxicity. Beetle body size and body condition index strongly and positively affected survival during monoterpene treatments. Larger beetles in better condition lost proportionally less mass during exposure, where proportion mass loss negatively affected survivorship. Toxicity was much more associated with water loss than with fat loss, suggesting that a main cost of detoxification is excretion, a process that has received little attention. These results provide insight into the determinants of beetle success in historic and novel hosts that differ in monoterpene composition and concentration. We also suggest that water availability will affect beetle success directly through their ability to tolerate detoxification as well as indirectly through host responses to drought.

Evaluation of teratogenic effects of crocin and safranal, active ingredients of saffron, in mice.

Saffron (Crocus sativus) is a widely used food additive for its color and taste. Crocin and safranal are two main components of this plant. Numerous studies are underway to introduce saffron and its active ingredients as pharmacological agents. Safety assessments of these compounds are important parts of this endeavor. In this study, the effects of crocin and safranal administrations during embryogenesis have been investigated in mice. A total of 75 BALB/c pregnant mice were divided into six experimental and control groups. Four experimental groups received intraperitoneal injection of crocin (200 mg/kg or 600 mg/kg) daily or safranal (0.075 ml/kg or 0.225 ml/kg) on gestational days (GDs) 6 to 15. Control groups received normal saline or paraffin as solvents of crocin and safranal. Dams were dissected on GD18 and embryos were collected. Routine maternal and fetal parameters were recorded. Macroscopic observation of external malformations was also performed. Fetuses were then selected for double skeletal staining with alizarin red and alcian blue. All experimental groups caused significant decrease in length and weight of fetuses when compared with the control groups and revealed malformations such as minor skeletal malformations, mandible and calvaria malformations, and growth retardation. Minor skeletal malformations were the most commonly observed abnormality, which were statistically significant when compared with the control groups (p < 0.05). The severities of malformations were comparable in the crocin- and safranal-treated groups. This study suggests that crocin or safranal can induce embryonic malformations when administered in pregnant mice. Due to the wide use of saffron, further elaborate studies to understand the malformation mechanisms of these ingredients are recommended.

Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC.

Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its potential as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain of Escherichia coli and found a mutation in ahpC, encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We show that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpC(L177Q)) was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We show that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-type E. coli cells. Our results have implications for both the renewable production of limonene and the applications of limonene as an antimicrobial.

Synthesis and biological activity of amino acid derivatives of avarone and its model compound.

A series of eighteen derivatives of marine sesquiterpene quinone avarone and its model system tert-butylquinone with amino acids has been synthesized by nucleophilic addition of amino acids to the quinones. In vitro cytotoxic activity toward human cancer cell lines (HeLa, A549, Fem-X, K562, MDA-MB-453) and normal MRC-5 cell line was determined. Several compounds showed very strong inhibitory activity with IC50 values less than 10 µM. Avarone derivatives were more active than the corresponding tert-butylquinone derivatives. The results of the cytofluorimetric analysis of cell cycle of HeLa cells showed that apoptosis might be one of possible mechanism of action of these compounds in cancer cells. In order to examine the influence of caspases on cell death, the apoptotic mechanisms induced by the tested compounds were determined using specific caspases 3, 8 and 9 inhibitors. For all compounds antibacterial activities against six strains of Gram-positive and four strains of Gram-negative bacteria were determined, as well as antifungal activity against three fungal species.

An assessment of anti-Müllerian hormone in predicting mating outcomes in female hamsters that have undergone natural and chemically-accelerated reproductive aging.

In mammals, female fertility declines with age due in part to a progressive loss of ovarian follicles. The rate of follicle decline varies among individuals making it difficult to predict the age of onset of reproductive senescence. Serum anti-Müllerian hormone (AMH) concentrations correlate with the numbers of ovarian follicles, and therefore, AMH could be a useful predictor of female fertility. In women and some production animals, AMH is used to identify which individuals will respond best to ovarian stimulation for assisted reproductive technologies. However, few studies have evaluated AMH's predictive value in unassisted reproduction, and they have yielded conflicting results. To assess the predictive value of AMH in the context of reproductive aging, we prospectively measured serum AMH in 9-month-old Siberian hamsters shortly before breeding them. Female Siberian hamsters experience substantial declines in fertility and fecundity by 9months of age. We also measured serum AMH in 5-month-old females treated with 4-vinylcyclohexene diepoxide (VCD), which selectively destroys ovarian follicles and functionally accelerates ovarian aging. Vehicle-treated 5-month-old females served as controls. AMH concentrations were significantly reduced in VCD-treated females yet many females with low AMH reproduced successfully. On average, both young and old hamsters that littered had higher AMH concentrations than females that did not. However, some females with relatively high AMH concentrations failed to litter, whereas several with low AMH succeeded. Our results suggest that mean AMH concentration can predict mating outcomes on a population or group level, but on an individual basis, a single AMH determination is less informative.

Conditions and possible mechanisms of VCD-induced ovarian failure.

Perimenopause is an important period in women's lives, in which they experience a series of physiological changes. Current animal models of perimenopause fail to adequately replicate this particular stage in female life, while current in vitro models are too simplistic and cannot account for systemic effects. Neither the naturally-ageing animal model, nor the ovariectomised animal model, mimic the natural transitional process that is the menopause. In vivo and in vitro studies have confirmed that the occupational chemical, 4-vinylcyclohexene diepoxide (VCD), can cause selective destruction of the ovarian primordial and primary follicles of rats and mice by accelerating the apoptotic process, which successfully mimics the perimenopausal state in women. However, it is the in vivo VCD-induced rodent perimenopausal models that are currently the most widely used in research, rather than any of the available in vitro models. Studies on the mechanisms involved have found that VCD induces ovotoxicity via interference with the c-kit/kit ligand and apoptotic signalling pathways, among others. Overall, the VCD-induced perimenopausal animal models have provided some insight into female perimenopause, but they are far from ideal models of the human situation.

Allergy to oxidized limonene and linalool is frequent in the U.K.

BACKGROUND: The oxidized forms of the fragrance terpenes limonene and linalool are known to cause allergic contact dermatitis. Significant rates of contact allergy to these fragrances have been reported in European studies and in a recent worldwide study. Patch testing to oxidized terpenes is not routinely carried out either in the U.K. or in other centres internationally. OBJECTIVES: To investigate the prevalence of contact allergy to oxidized limonene and linalool in the U.K. METHODS: Between 1 August 2011 and 31 December 2012, 4731 consecutive patients in 13 U.K. dermatology departments were tested for hydroperoxides of limonene 0·3% pet., hydroperoxides of linalool 1·0% pet., stabilized limonene 10·0% pet. and stabilized linalool 10·0% pet. Doubtful (?+) and equivocal (±) reactions were grouped together as irritant reactions. RESULTS: Two hundred and thirty-seven patients (5·0%) had a positive patch test reaction to hydroperoxides of limonene 0·3% pet. and 281 (5·9%) to hydroperoxides of linalool 1·0% pet. Irritant reactions to one or both oxidized terpenes were found in 242 patients (7·3%). Eleven patients (0·2%) had a positive patch test reaction to the stabilized terpenes alone. CONCLUSIONS: This large, multicentre U.K. audit shows a significant rate of allergy to the hydroperoxides of limonene and linalool plus a high rate of irritant reactions. Testing to the oxidized forms alone captures the majority (97·0%; 411 of 422) of positive reactions; testing to nonoxidized terpenes appears to be less useful. We recommend that the hydroperoxides of limonene and linalool be added to an extended baseline patch test series.

Assessment of the risk of respiratory sensitization from fragrance allergens released by air fresheners.

Consumers using air fresheners are exposed to the emitted ingredients, including fragrances, via the respiratory tract. Several fragrances are known skin sensitizers, but it is unknown whether inhalation exposure to these chemicals can induce respiratory sensitization. Effects on the immune system were assessed by testing a selection of five fragrance allergens in the respiratory local lymph node assay (LLNA). The probability and extent of exposure were assessed by measuring concentrations of the 24 known fragrance allergens in 109 air fresheners. It was shown that the most frequently used fragrances in air fresheners were D-limonene and linalool. In the respiratory LLNA, these fragrances were negative. Of the other tested chemicals, only isoeugenol induced a statistically significant increase in cell proliferation. Consumer exposure was assessed in more detail for D-limonene, linalool, and isoeugenol by using exposure modeling tools. It was shown that the most frequently used fragrances in air fresheners, D-limonene, and linalool gave rise to a higher consumer exposure compared with isoeugenol. To evaluate whether the consumer exposure to these fragrances is low or high, these levels were compared with measured air concentrations of diisocyanates, known human respiratory sensitizers. This comparison showed that consumer exposure from air fresheners to D-limonene, linalool, and isoeugenol is considerably lower than occupational exposure to diisocyanates. By combing this knowledge on sensitizing potency with the much lower exposure compared to diisocyanates it seems highly unlikely that isoeugenol can induce respiratory sensitization in consumers using air fresheners.

Glutathione S-transferase class µ regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries.

4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 µM) for 2-8 days; 2) VCD (30 µM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 µM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P<0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P<0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P<0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P<0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1.

Safety evaluation and risk assessment of d-Limonene.

d-Limonene, a major constituent of citrus oils, is a monoterpene widely used as a flavor/fragrance additive in cosmetics, foods, and industrial solvents as it possesses a pleasant lemon-like odor. d-Limonene has been designated as a chemical with low toxicity based upon lethal dose (LD50) and repeated-dose toxicity studies when administered orally to animals. However, skin irritation or sensitizing potential was reported following widespread use of this agent in various consumer products. In experimental animals and humans, oxidation products or metabolites of d-limonene were shown to act as skin irritants. Carcinogenic effects have also been observed in male rats, but the mode of action (MOA) is considered irrelevant for humans as the protein α(2u)-globulin responsible for this effect in rodents is absent in humans. Thus, the liver was identified as a critical target organ following oral administration of d-limonene. Other than the adverse dermal effects noted in humans, other notable toxic effects of d-limonene have not been reported. The reference dose (RfD), the no-observed-adverse-effect level (NOAEL), and the systemic exposure dose (SED) were determined and found to be 2.5 mg/kg/d, 250 mg/kg//d, and 1.48 mg/kg/d, respectively. Consequently, the margin of exposure (MOE = NOAEL/SED) of 169 was derived based upon the data, and the hazard index (HI = SED/RfD) for d-limonene is 0.592. Taking into consideration conservative estimation, d-limonene appears to exert no serious risk for human exposure. Based on adverse effects and risk assessments, d-limonene may be regarded as a safe ingredient. However, the potential occurrence of skin irritation necessitates regulation of this chemical as an ingredient in cosmetics. In conclusion, the use of d-limonene in cosmetics is safe under the current regulatory guidelines for cosmetics.

Exogenous ergosterol protects Saccharomyces cerevisiae from D-limonene stress.

AIMS: Enhancement of the tolerance of Saccharomyces cerevisiae to monoterpenes has the potential to improve the de novo biosynthesis of these chemicals as well as the efficient utilization of monoterpene-containing citrus waste. The aims of the current work are to demonstrate the mechanisms by which ergosterol, an important component of cell membranes, protects S. cerevisiae from D-limonene stress and to provide some useful information for further metabolic engineering of the yeast. METHODS AND RESULTS: Saccharomyces cerevisiae cells were treated with a sublethal dose of D-limonene for 2 h, and then ergosterol was added to investigate the physiological responses of S. cerevisiae. In D-limonene-treated cells, the membrane fluidity, permeability and saturated fatty acid ratio increased, whereas the intracellular ergosterol concentration decreased sharply. Addition of ergosterol restored membrane and intracellular ergosterol to normal levels. Exogenous ergosterol triggered nearly all of the genes that encode the biosynthesis of ergosterol. CONCLUSIONS: In S. cerevisiae, the cell membrane is the target of D-limonene. Intracellular ergosterol availability is correlated with the D-limonene tolerance of the cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that modification of the ergosterol biosynthesis pathway could be a promising strategy for constructing a robust yeast strain with enhanced tolerance.

Adjuvant and inflammatory effects in mice after subchronic inhalation of allergen and ozone-initiated limonene reaction products.

Inhalation of ozone (O3), a highly toxic environmental pollutant, produces airway inflammation and exacerbates asthma. However, in indoor air, O3 reacts with terpenes (cyclic alkenes), leading to formation of airway irritating pollutants. The aim of the study was to examine whether inhalation of the reaction products of O3 and the terpene, limonene, as well as limonene and low-level O3 by themselves, induced allergic sensitization (formation of specific immunoglobulin [Ig] E) and airway inflammation in a subchronic mouse inhalation model in combination with the model allergen ovalbumin (OVA). BALB/cJ mice were exposed exclusively by inhalation for 5 d/wk for 2 wk and thereafter once weekly for 12 wk. Exposures were low-dose OVA in combination with O3, limonene, or limonene/O3 reaction products. OVA alone and OVA + Al(OH)3 served as control groups. Subsequently, all groups were exposed to a high-dose OVA solution on three consecutive days. Serum and bronchoalveolar lavage fluid were collected 24 h later. Limonene by itself did not promote neither OVA-specific IgE nor leukocyte inflammation. Low-level O3 promoted eosinophilic airway inflammation, but not OVA-specific IgE formation. The reaction products of limonene/O3 promoted allergic (OVA-specific IgE) sensitization, but lung inflammation, which is a characteristic of allergic asthma, was not observed. In conclusion, the study does not support an allergic inflammatory effect attributed to O3-initiated limonene reaction products in the indoor environment.

Maternal exposure to 4-vinylcyclohexene diepoxide during pregnancy induces subfertility and birth defects of offspring in mice.

4-vinylcyclohexene diepoxide (VCD), widely used in industry, is a hazardous compound that can cause premature ovarian failure, but whether maternal VCD exposure affects the health and reproduction of offspring is unknown. Here we focused on the effects of VCD on fertility and physical health of F1 and F2 offspring in mice. The pregnant mice were injected intraperitoneally with different dosages of VCD once every day from 6.5 to 18.5 days post-coitus (dpc). We showed that maternal exposure to VCD during pregnancy significantly reduced the litter size and ovarian reserve, while increasing microtia occurrences of F1 mice. The cytospread staining showed a significant inhibition of meiotic prophase I progression from the zygotene stage to the pachytene stage. Mechanistically, the expression level of DNA damage marker (γ-H2AX) and BAX/BCL2 ratios were significantly increased, and RAD51 and DMC1 were extensively recruited to DNA double strand breaks sites in the oocytes of offspring from VCD-exposed mothers. Overall, our results provide solid evidence showing that maternal exposure to VCD during pregnancy has intergenerational deleterious effects on the offspring.

4-vinylcyclohexene diepoxide induces apoptosis by excessive reactive oxygen species and DNA damage in human ovarian granulosa cells.

4-Vinylcyclohexene diepoxide (VCD) is a hazardous industrial material which is widely used in the production of fragrances, rubber tires, antioxidants, pesticides, flame retardants and plasticizers. Previous studies have shown that exposure to VCD damages the female reproductive system, but the effects and mechanisms of VCD exposure on human granulosa cells are not reported. In this study, we used a human granulosa cell line (SVOG) to explore the effects of VCD exposure and found that VCD exposure had toxic effects on SVOG cells in vitro. VCD exposure led to excessive accumulation of intracellular ROS, caused DNA damage in cells, altered the expression of some key genes related with apoptosis and oxidative stress, and ultimately inhibited the proliferative capacity of granulosa cells, resulting in increased apoptosis. Overall, our findings provide solid evidence showing that VCD exposure produces severe damage to human granulosa cells, which is helpful for understanding the reproductive toxicity of VCD and etiology of infertility.