Paternal exposure to di-n-butyl-phthalate induced developmental toxicity in zebrafish (Danio rerio).

BACKGROUND: Dibutyl phthalate (DBP) is an environmental endocrine disruptor detected in water, soil, and other environmental media frequently. Growing concerns regarding DBP exposure focus on toxicity to male reproduction. Reports about the developmental toxicity of paternal DBP exposure are rare. In this study, we investigated the developmental toxicity of paternal exposure to DBP on offspring in zebrafish. METHODS: Adult male zebrafish with normal reproductive function were exposed to 0.2, 0.6, 1.8 mg/L of DBP or acetone solvent control for 30 days, and then mated with females. Thirty embryos per group were randomly selected to be observed, and malformations were recorded and photographed. The mating and observations were repeated three times, for a total of 90 embryos per group. RESULTS: The results showed that the percentage of malformations, such as edema and a bent trunk, was increased in the 0.6 and 1.8 mg/L DBP exposure groups, the heart rate and spontaneous contraction decreased in the 0.6 and 1.8 mg/L DBP exposure groups and migration of primordial germ cells was disrupted in some F1 embryos in all DBP exposure group after paternal exposure. The axial skeleton was affected in some F1 adults in the 1.8 mg/L DBP exposure group. CONCLUSIONS: Our findings demonstrate the developmental toxicity of paternal DBP exposure in zebrafish.

dl-Mandelic acid exhibits high sperm-immobilizing activity and low vaginal irritation: A potential non-surfactant spermicide for contraception.

dl-Mandelic acid (MA), an alpha-hydroxycarboxylic acid, has been widely used as an intermediate of pharmaceutical and fine chemicals. Here, we evaluated the sperm-immobilizing activity of MA and its safety profiles. Spermatozoon motility was assessed by computer-aided sperm analysis, the integrity of the plasma membrane and. mitochondrial potential was assessed using fluorescein isothiocyanate-pisum sativum agglutinin and JC-1, respectively. The local tolerance of the MA-containing gel formulation was evaluated using a rabbit vaginal irritation test. We found that MA inhibited sperm motility and movement patterns in a concentration-dependent manner. Within 20 s, MA-induced spermatozoa immobilization occurred with a minimum effective concentration and a median effective concentration of 0.86 and 0.54 mg/mL, respectively. Plasma membrane disruptions of MA-treated spermatozoa were relatively mild, but mitochondrial depolarization occurred. Histopathological examination showed that MA exposure did not exert obvious effects on the integrity of spermatozoa membrane structures and only caused slight irritation to the rabbit vaginal epithelium. The vaginal irritation scores of the vehicle control and the nonoxynol -9 gel control groups were 1.38 ± 0.65 and 7.88 ± 1.67, respectively (p < 0.01), whereas those of the MA gel groups at 10, 20, and 40 mg/mL were 1.69 ± 1.04, 2.98 ± 0.77, and 4.35 ± 1.04 with p values of >0.05, >0.05, and <0.05 (vs. vehicle control), respectively, which were within the clinically acceptable range (<8). Therefore, our results confirmed that MA exhibited significant sperm-immobilizing effects and caused mild plasma membrane injury, suggesting that it has potential for development as a future non-surfactant spermicide.

Continuing harmonization of terminology and innovations for methodologies in developmental toxicology: Report of the 8th Berlin Workshop on Developmental Toxicity, 14-16 May 2014.

This article is a report of the 8th Berlin Workshop on Developmental Toxicity held in May 2014. The main aim of the workshop was the continuing harmonization of terminology and innovations for methodologies used in the assessment of embryo- and fetotoxic findings. The following main topics were discussed: harmonized categorization of external, skeletal, visceral and materno-fetal findings into malformations, variations and grey zone anomalies, aspects of developmental anomalies in humans and laboratory animals, and innovations for new methodologies in developmental toxicology. The application of Version 2 terminology in the DevTox database was considered as a useful improvement in the categorization of developmental anomalies. Participants concluded that initiation of a project for comparative assessments of developmental anomalies in humans and laboratory animals could support regulatory risk assessment and university-based training. Improvement of new methodological approaches for alternatives to animal testing should be triggered for a better understanding of developmental outcomes.

Effectiveness of an optimized benzalkonium chloride gel as vaginal contraceptive: a randomized controlled trial among Chinese women.

BACKGROUND: This study was conducted to evaluate the efficacy, safety and acceptability of a newly developed benzalkonium chloride (BZK) contraceptive gel which was compared to nonoxynol-9 (N-9) gel. STUDY DESIGN: A Phase II, multicenter, randomized, controlled study at three Chinese centers was conducted to compare 120 women who used BZK gel with 120 women who used N-9 gel for 6 months. Contraceptive efficacy was assessed by pregnancy rate, and safety was evaluated by adverse events report, gynecologic examination, Papanicolaou smears, leukorrhea test, and blood and urine tests. The acceptability was assessed through follow-up visit forms and a questionnaire at the 6-month visit. RESULTS: Net cumulative rates in the BZK group at 6 months were as follows: follow-up 100%, terminations 5.1%, pregnancy 1.7%, medical reasons 0% and fear of failure 3.4%. At 6 months, the rates in the N-9 group were as follows: follow-up 99.2%, terminations 9.4%, pregnancy 0.9%, medical reasons 2.5%, fear of failure 3.4% and other personal reasons 2.6%. No significant difference in pregnancy rate and termination rate between the two groups was found (p>.05). Seven cases in the BZK group (5.8%) complained about leukorrhagia and vaginal irritation symptoms (itching and burning) at 6 months, while 16 cases in the N-9 group (13.3%) had similar complaints (p<.05). This significant difference continued to exist until the 6-month visit. The general satisfaction rate for BZK gel use (72.8%) is significantly higher than that for N-9 gel (42.5%). CONCLUSION: The optimized BZK gel is comparable to N-9 gel in terms of contraceptive efficacy and safety, and may be more acceptable to Chinese users.

[Exposure to 1-bromopropane causes dose-dependent neurological abnormalities in workers].

OBJECTIVE: To explore the dose-effect relationship between 1-bromopropane (1-BP) exposure and health effects in workers. METHODS: Occupational field investigations were conducted in 1-BP factories. Ambient 1-BP concentrations were detected with detection tube, and the 8 h time-weighted average individual exposure levels (TWA-8 h) were measured by passive sampler. Workers underwent questionnaire survey, neurological examination, nerve conduction velocity examination, vibration sensation test. routine blood test as well as blood biochemical test. According to TWA values or TWA x duration values, workers were divided into three dose groups for dose-effect relationship analysis. USEPA BMDS 2.1 software was applied to calculate 1-BP benchmark dose (BMD) and its 95% lower limit (BMDL). RESULTS: The TWA-8h concentrations ranged from 0.35 to 535.19 mg/m3 (geo-mean 14.08 mg/m3). Dose-dependent analysis showed that the motor nerve distal latency (linear regression coefficient was 0.066 6), vibration sensation of toes (linear regression coefficient were 0.157 2 and 0.193 9), creatine kinase (linear regression coefficient was -1.05) and thyroid stimulating hormone levels (linear regression coefficient was 0.1024) of 1-BP exposed workers changed in a dose-dependent manner (P < 0.05). BMD calculation based on DL as 1-BP toxic effect endpoint showed that TWA-8h of the BMD values and BMDL values were 50.55 mg/m3 and 30.78 mg/m3, respectively. CONCLUSION: 1-BP causes dose-dependent changes in tibial nerve DL, vibration sensation, CK and TSH levels.

[Effects of 1-bromopropane on neurological and hematological changes of female exposed workers].

OBJECTIVE: To investigate the health effects of 1-bromopropane (1-BP) on female exposed workers. METHODS: Four 1-BP manufacturing plants were investigated. Workers were interviewed with questionnaire and examined with neurobehavioral core test battery, nerve conduction velocity tests of nervus tibialis and nervus suralis, vibration sensation test, hematological and biochemical tests. Ambient 1-BP concentration was measured with detection tube, and time-weighed average levels of individual workers were estimated with passive samplers. RESULTS: 1-BP concentration in the plants ranged from 0 to 402.40 mg/m3 (Geomean 32.19 mg/m3). Time-weighted average exposure levels (TWA-8 h) ranged from 0.35 to 535.19 mg/m3 (Geomean 14.08 mg/m3). Compared with the control group, 1-BP exposed workers showed reduced motor nerve conduction velocity [(44.8 +/- 8.7) m/s] and sensory nerve conduction velocity [(45.5 +/- 4.9) m/s], prolonged distal latency [(7.5 +/- 2.1) ms], reduced toe vibration perception, and altered neurobehavior parameters(POMS vigor, tension, anxiety, confusion) significantly (P < 0.05). As to hematological and biochemical indicators, the exposed workers showed decreased white blood cell count [(5.6 +/- 2.17) x 10(3)/microl], red blood cell count [(3.9 +/- 0.4) x 10(6)/microl], hemoglobin [(121.1 +/- 14.5) g/L] and creatine kinase [(82.0 +/- 27.5) IU/L] (P < 0.05), and increased serum total protein (8.0 +/- 0.5 g/dl), lactate dehydrogenase [(335.2 +/- 356.6) IU/L], thyroid-stimulating hormone [(3.6 +/- 2.3) microIU/ml] and follicle-stimulating hormone levels (18.7 +/- 24.4 mIU/ml) (P < 0.05). CONCLUSION: 1-BP exposure may affect peripheral nerves and central nervous system, and lead to abnormal hematological and biomedical indicators.

Dose-dependent neurologic abnormalities in workers exposed to 1-bromopropane.

OBJECTIVES: To investigate the health effects of 1-bromopropane (1-BP) and its dose-dependency in 1-BP production factories in China. METHODS: Data of 60 female and 26 male workers in three 1-BP factories and the same number of age-, sex-, and region-matched controls were interviewed and examined. The time-weighed average exposure levels of individual workers were estimated. RESULTS: Regression analysis on exposure level showed dose-dependent increase in the distal latency of tibial nerve, threshold for vibration sense in toes, lactate dehydrogenase, thyroid stimulating hormone, and follicle stimulating hormone in female workers. The analysis also showed dose-dependent decrease in sensory nerve conduction velocity of the sural nerve, red blood cell, and hematocrit in female workers. CONCLUSIONS: The results indicate that exposure to 1-BP induces dose-dependent neurotoxicity in female workers.

[Change of gonad gene expression profile in male F344 rats after exposure to 1-bromopropane].

OBJECTIVE: To study the 1-bromopropane (1-BP)-induced altered gene expression profiles in rat gonad, and explore its male reproductive toxicity-related mRNA changes. METHODS: Twelve F344/NSIc male rats were randomly divided into two groups of 6 each. Rats were exposed to either fresh air or 5030 mg/m3 1-BP through inhalation for 8 h. Rats were sacrificed and testes were removed at 16 h after exposure. Global changes in gene expression were determined by microarray analysis using rat genital chip followed by Real-time PCR validation. RESULTS: Among the 5082 genes and ESTs in the genital chip, 62 genes were down-regulated and 3 genes were up-regulated by 1-BP, which include synthetic sex hormone-related genes cytochrome P450 aromatase (CYP19a), glutathione S-transferase (GSTT1), creatine kinase (Ckb), myelin and lymphocyte protein (Mal) and S100 calcium-binding protein (S100a4). Classification analysis revealed that the majority of gene changes was involved protein/lipid metabolism, followed by the stress-associated defense response. Real-time PCR confirmed the down-regulation of CYP19a, GSTT1, Mal and S100a4 genes. CONCLUSION: Acute high-dose exposure to 1-BP causes the down-regulation of testicular CYP19a, S100a4, GSTT1 and Mal. This altered gene profiles might reflect the toxic mechanism which suggested that 1-BP disrupt the metabolics and endocrine balance.

[Study on gonadal developmental toxicity of dibutyl phthalate in male zebrafish of F1 generation].

OBJECTIVE: This study aims to explore the effects of dibutyl phthalate (DBP) on the gonad development of F1 generation male zebrafish. METHODS: Fourty-eight pairs of adult zebrafish were randomly divided into 4 groups of 12 pairs each: blank control group, 0.01% acetone solvent control group, 1250 microg/L DBP high-dose group, and 625 microg/L DBP low-dose group. After 30 days of exposure, zebrafish in each group were mated and the F1 generations were raised up to 180 dph in clean water. Twenty F1 male zebrafish in each group underwent gonadal index (GSI) analysis and gonad histopathology examination. RESULTS: GSI of F1 male zebrafish in high-dose group (5.23% +/- 1.5%) was significantly lower than that in blank control (6.09% +/- 2.5%) or solvent control (6.08% +/- 1.2%) (P < 0.05). Histological examination on testis showed that DBP inhibited male gonadal development of the F1 offspring and led to abnormal seminiferous tubules with clear characteristics of extensive and significant proliferation of Leydig cells accompanied by low sperm count. CONCLUSION: Gonadal development can be inhibited to different degrees by prenatal exposure to DBP in F1 male zebrafish, including abnormal seminiferous tubules, reduced sperm count as well as Leydig cell hyperplasia. This anti-estrogen activity of DBP in zebrafish is similar to those DBP-induced testicular effects observed in male rats, marmoset and human beings.

[Embryotoxicity of di-n-butyl phthalate to zebrafish (Brchydanio rerio) embryos].

OBJECTIVE: To investigate the embryotoxicity of Di-n-butyl phthalate (DBP) on zebrafish. METHODS: In this study, 80 healthy adult zebrafish were selected, containing 40 female and 40 male. After separated raised for one month, they were separated 4 groups. Each group was exposed to 1250 microg/L DBP(dissolved in 0.01% acetone), 625 microg/L DBP (dissolved in 0.01% acetone), 0.01% acetone and water, respectively. Acetone and water were used as solvent control and blank control, respectively. After exposed to DBP for seven days, zebrafish were mated at the ratio of 1 : 1 (male to female). Embryo development, fertility rate, 72h death rate, 72h hatching ratio, body weight and length of zebrafish were observed under the inverted microscope and by using microbalance and callipers. RESULTS: The fertility rates of the blank group, acetone control group, 625 microg/L DBP-treated group and 1250 microg/L DBP-treated group were 98.09%, 95.76%, 95.31% and 94.42%, respectively. And the 72h death rates of various groups were 18.29%, 23.34%, 49.45% and 72.41%, respectively. The hatching ratios were 96.43%, 82.37%, 48.11% and 26.79%. The 72h death rates of DBP-treated groups were higher than those of non-treated groups, but the hatching ratios were lower. Compared with the blank and acetone control group, retardation of embryo development in DBP-treated groups were observed obviously. Although no significant differences were observed on body weight, the body length were significant shorter in DBP-treated groups, while comparing with blank and acetone control group. The higher dose zebrafish exposed, the less body length the young had. CONCLUSION: 625 microg/L and higher DBP treated may be the cause of the retardation of zebrafish embryo development.

Globin s-propyl cysteine and urinary N-acetyl-S-propylcysteine as internal biomarkers of 1-bromopropane exposure.

1-Bromopropane (1-BP), an alternative to ozone-depleting solvents, is a neuro and reproductive toxicant in animals and humans. In this study, the dose responses for urinary AcPrCys and S-propylcysteine (PrCys) adducts on globin and neurofilaments were determined as a function of 1-BP exposure level and duration in the rat; and globin PrCys adducts and urinary AcPrCys were quantified in samples obtained from workers in a 1-BP production facility. Rats were exposed to 1-BP by inhalation for 2 weeks at 0, 50, 200, or 800 ppm and to 1-BP at 0 or 50 ppm for 4 weeks. After the 4-week exposures ended, half of the animals were euthanized immediately and half euthanized 8 days later. Urinary AcPrCys was measured using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and gas chromatograph-mass spectrometry (GC/MS); and PrCys adducts were determined on globin and neurofilaments using LC/MS/MS. In rats, PrCys adduct and urinary AcPrCys levels demonstrated a linear dose response relative to exposure level. PrCys globin adducts demonstrated a linear cumulative dose response over the 4-week exposure period. Elimination of AcPrCys appeared biphasic with detectable levels still present in urine up to 8 days postexposure. A significant increase in globin PrCys adducts was observed in the 1-BP workers relative to control workers; and urinary AcPrCys increased with increasing 1-BP ambient exposure levels. The results of these studies demonstrate the ability of 1-BP to covalently modify proteins in vivo and support the potential of urinary AcPrCys and globin PrCys adducts to serve as biomarkers of 1-BP exposure in humans.

Neurologic abnormalities in workers of a 1-bromopropane factory.

We reported recently that 1-bromopropane (1-BP; n-propylbromide, CAS Registry no. 106-94-5), an alternative to ozone-depleting solvents, is neurotoxic and exhibits reproductive toxicity in rats. The four most recent case reports suggested possible neurotoxicity of 1-BP in workers. The aim of the present study was to establish the neurologic effects of 1-BP in workers and examine the relationship with exposure levels. We surveyed 27 female workers in a 1-BP production factory and compared 23 of them with 23 age-matched workers in a beer factory as controls. The workers were interviewed and examined by neurologic, electrophysiologic, hematologic, biochemical, neurobehavioral, and postural sway tests. 1-BP exposure levels were estimated with passive samplers. Tests with a tuning fork showed diminished vibration sensation of the foot in 15 workers exposed to 1-BP but in none of the controls. 1-BP factory workers showed significantly longer distal latency in the tibial nerve than did the controls but no significant changes in motor nerve conduction velocity. Workers also displayed lower values in sensory nerve conduction velocity in the sural nerve, backward recalled digits, Benton visual memory test scores, pursuit aiming test scores, and five items of the Profile of Mood States (POMS) test (tension, depression, anxiety, fatigue, and confusion) compared with controls matched for age and education. Workers hired after May 1999, who were exposed to 1-BP only (workers hired before 1999 could have also been exposed to 2-BP), showed similar changes in vibration sense, distal latency, Benton test scores, and depression and fatigue in the POMS test. Time-weighted average exposure levels in the workers were 0.34-49.19 ppm. Exposure to 1-BP could adversely affect peripheral nerves or/and the central nervous system.

[Reproductive and developmental toxicity of F1 male rats treated with DBP in utero and during lactation].

OBJECTIVE: To observe the effects of Di-n-butyl phthalate (DBP) on the developmental condition of F1 rats and on the reproductive system of mature F1 male rats and to establish the NOAEL of DBP. METHODS: In this study, pregnant rats were treated with different dose of DBP (0, 50, 250 and 500 mg per kg per day) by gavage in utero and during lactation (from GD1 to PND21). The effects of DBP on F1 rats were observed. RESULTS: DBP had no obvious effect on pregnant rats but could decline pups' born weight, live pups per litter, body weight gain and male pups' anogenital distance obviously. Severe damages on the reproductive system of mature F1 male rats (testicular atrophy, underdevelopmental epididymis, absent of epididymis, undescended testes, obvious decline of epididymal sperm parameters, total sperm heads per g testis, decrease of organ/body weight ratio of epididymis, liver, kidney and prostate, etc.) were observed in the group of 250 mg per kg per day and higher. CONCLUSION: These results showed that male reproductive system was the target organ of DBP and the damages on pups were partly irreversible. According to these results, the NOAEL of DBP, which was not available in the current database, was established based on the outcomes of this study, i.e., 50 mg per kg per day. Accordingly, the RfD for human exposure to DBP through oral intake was recommended as 500 micrograms per kg per day.

A survey on exposure level, health status, and biomarkers in workers exposed to 1-bromopropane.

BACKGROUND: The aim of this study was to evaluate the health effects of exposure mainly to 1-bromopropane, which is an alternative to ozone-depleting solvents, and to establish biomarkers for assessing 1-bromopropane exposure. METHODS: Twenty-four female and 13 male workers of a 1-bromopropane-factory were interviewed, and their urine and blood samples were collected. Measured parameters included 1-bromopropane levels in the factory, as well as individual exposure levels, urinary 1-bromopropane levels, enzymatic activity and M subunit's concentration of serum creatine kinase (CK). RESULTS: Frequent symptoms reported by workers exposed to 1-bromopropane were nose, throat, and eyes irritation or malaise and/or headache. Urinary 1-bromopropane levels correlated significantly with individual exposure levels, but enzymatic activity or CK-M subunit did not. CONCLUSIONS: The symptoms suggested irritation of the mucous membrane and possible adverse effects on the central nervous system. There were no severe chronic symptoms suggestive of neurological damage in workers exposed to less than 170 ppm. Urinary 1-bromopropane level may be a good indicator of exposure. Am. J. Ind. Med. 45:63-75, 2004.

2-Bromopropane induces DNA damage, impairs functional antioxidant cellular defenses, and enhances the lipid peroxidation process in primary cultures of rat Leydig cells.

Utilization of highly enriched preparations of steroidogenic Leydig cells has proven invaluable for studying the direct effects of various hormones and agents on Leydig cell function in vitro. It is widely reported that male reproductive organs are particularly susceptible to the deleterious effects of reactive oxygen species (ROS) and lipid peroxidation, which ultimately lead to impaired fertility. The purpose of the study was to examine the potential of 2-bromopropane (2-BP) to induce oxidative stress and antioxidant function in primary cultures of rat Leydig cells. Leydig cells were isolated from the testes of Sprague-Dawley rats. The purity of Leydig cells was determined to be 94.6% and the cells maintained their testosterone secreting capabilities for 48 h. Fresh medium containing 2-BP (1.00, 0.10, 0.01 mM, and vehicle control) and 1 U human chorionic gonadotropin (hCG) were added in the cell culture. Superoxide dismutase (SOD) activity, malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) were analyzed in the medium of each well by biochemical methods. Additionally, DNA damage was examined using the Comet assay. The proportion of cells with undamaged DNA was decreased significantly and those with different grades of damaged DNA were increased significantly in the cells exposed to 2-BP. The level of MDA and GSH-PX activity increased significantly in the cell groups exposed to 0.10 and 1.00 mM 2-BP, whereas, SOD activity decreased considerably in these two groups of cells when compared to the control. The data indicate that 2-BP induces DNA damage, impairs functional antioxidant cellular defenses, and enhances the lipid peroxidation in cultured Leydig cells. These effects may be responsible for the testicular toxicity noted in laboratory animals and humans.