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There are currently no skin sensitization assays based on T cell activation. We built a novel in vitro model to assess T cell activation and test its performance to discriminate skin sensitizers from non-sensitizers using 52 reference chemicals. Jurkat Clone E6-1 human T lymphocytes were exposed to a series of concentrations of test substances for 24 hours and CD69 expression was measured as a marker of early T cell activation with flow cytometry. Most tested sensitizers induced increased relative fluorescence intensity (RFI) of CD69 on the T cells, which was linearly correlated with the concentrations tested, indicating a statistically significant causal link between sensitizer concentration and increase of CD69 expression. CD69 RFI ≥ 1.5 was determined as the positive criterion for skin sensitizer classification. The sensitivity (79.4%), specificity (88.9%) and accuracy (82.7%) of the model for the 52 tested reference chemicals showed a good predictivity for skin sensitizers. The results were reproduced in at least two repeats; and the concurrent positive control, 2 mg/mL 2, 4-dinitrochlorobenzene, was found positive in all 25 independent runs conducted, indicating in-house reproducibility. The EC1.5 value (i.e., the concentration at which a test chemical induces a CD69 RFI of 1.5) may be used to assess skin sensitization potency of a chemical. This work may contribute to the development of an in vitro assay for skin sensitization based on the activation of T cells.

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What is already known on this topic? Starting in the early 1950s, the main industries in China associated with chronic benzene poisoning (CBP) included painting, pharmaceuticals, and shoemaking. However, because of rapid socioeconomic development, the distribution of industries associated with CBP likely changed. What is added by this report? From 2005 to 2019, CBP has become an increasingly important type of chronic occupational poisoning (COP) in China. CBP was mainly found to have occurred in manufacturing industries, especially private enterprises and small and medium-sized enterprises. The sub-industry with the highest proportion of CBP cases was general and special equipment manufacturing, followed by chemical raw materials and chemical manufacturing. What are the implications for public health practice? CBP was found to be the main component of COP in China, so the supervision and management in manufacturing, especially in the medium-sized and small enterprises, need to be strengthened. Occupational benzene exposure limits should also be adjusted accordingly.

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Benzene, a widespread environmental pollutant, induces DNA double-strand breaks (DSBs) and DNA repair, which may further lead to oncogenic mutations, chromosomal rearrangements and leukemogenesis. However, the molecular mechanisms underlying benzene-induced DNA repair and carcinogenesis remain unclear. The human osteosarcoma cell line (U2OS/DR-GFP), which carries a GFP-based homologous recombination (HR) repair reporter, was treated with hydroquinone, one of the major benzene metabolites, to identify the potential effects of benzene on DSB HR repair. RNA-sequencing was further employed to identify the potential key pathway that contributed to benzene-initiated HR repair. We found that treatment with hydroquinone induced a significant increase in HR. NF-κB pathway, which plays a critical role in carcinogenesis in multiple tumors, was significantly activated in cells recovered from hydroquinone treatment. Furthermore, the upregulation of NF-κB by hydroquinone was also found in human hematopoietic stem and progenitor cells. Notably, the inhibition of NF-κB activity by small molecule inhibitors (QNZ and JSH-23) significantly reduced the frequency of hydroquinone-initiated HR (-1.36- and -1.77-fold, respectively, P < 0.01). Our results demonstrate an important role of NF-κB activity in promoting HR repair induced by hydroquinone. This finding sheds light on the underlying mechanisms involved in benzene-induced genomic instability and leukemogenesis and may contribute to the larger exploration of the influence of other environmental pollutants on carcinogenesis.

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Benzene is a primary industrial chemical and a ubiquitous environmental pollutant. ERCC3 is a key player in nucleotide excision repair. Recent studies suggested that site-specific methylation is a possible mechanism of the transcriptional dysregulation by blocking transcription factors binding. We previously found that the average promoter methylation level of ERCC3 was increased in benzene-exposed workers. In order to test whether specific CpG sites of ERCC3 play an important role in benzene-induced epigenetic changes and whether the specific methylation patterns are associated with benzene hematotoxicity, we analyzed the promoter methylation levels of individual CpG sites, transcription factor binding motif and the correlation between aberrant CpG methylation and hematotoxicity in 76 benzene-exposed workers and 24 unexposed controls in China. Out of all the CpGs analyzed, two CpG units located 43 bp upstream and 99 bp downstream of the transcription start site of ERCC3 (CpG 2-4 and CpG 17-18, respectively), showed the most pronounced increase in methylation levels in benzene-exposed workers, compared with unexposed controls (Mean ± SD: 5.86 ± 2.77% vs. 4.92 ± 1.53%, p = 0.032; 8.45 ± 4.09% vs. 6.79 ± 2.50%, p = 0.024, respectively). Using the JASPAR CORE Database, we found that CpG 2-4 and CpG 17-18 were bound by three putative transcription factors (TFAP2A, E2F4 and MZF1). Furthermore, the methylation levels for CpG 2-4 were correlated negatively with the percentage of neutrophils (ß = -0.676, p = 0.005) in benzene-exposed workers. This study demonstrates that CpG-specific DNA methylation in the ERCC3 promoter region may be involved in benzene-induced epigenetic modification and it may contribute to benzene-induced hematotoxicity.

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INTRODUCTION: The Local Lymph Node Assay (LLNA) has been designated as the first-choice in vivo assay for identification the skin sensitization potential of new chemicals. The LLNA:BrdU-ELISA is a validated non-radioactive modification to the LLNA. An intra-laboratory reproducibility study for the LLNA:BrdU-ELISA was conducted to demonstrate its adequate performance in our laboratory. METHODS: Ten independent LLNA:BrdU-ELISAs with the preferred positive controls (PCs), i.e., 25% hexyl cinnamic aldehyde (HCA) and 25% eugenol, were conducted within a period of less than one year. In addition, different concentrations of 2,4-dinitrochlorobenzene (DNCB, an extreme sensitizer) (0.01, 0.1 and 0.3%), HCA (10, 25 and 50%) and eugenol (10, 25 and 50%), were tested to determine the EC1.6 values. Special Pathogen Free female CBA/J mice of 8-10weeks old were randomly allocated to the groups, each group having 4 mice. 25µl of AOO (vehicle, acetone: olive oil=4:1, v/v) or HCA, eugenol, DNCB at the needed concentration was applied to the dorsum of each ear of the mice, daily for 3 consecutive days. A single intraperitoneal injection of 0.5ml of BrdU solution (10mg/ml) was given on day 5. On day 6, a pair of auricular lymph nodes from each mouse was excised, and BrdU ELISA analysis was conducted. RESULTS: The result for each group is expressed as the mean Stimulation Index (SI). The mean of the 10 mean SIs for 25% HCA (2.58±0.95) and 25% eugenol (3.51±1.25) was not significantly different to that from the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) (i.e., the data on the formal validation study for the LLNA:BrdU-ELISA by the ICCVAM) (3.03±2.00 for 25% HCA, 6.13±6.06 for 25% eugenol) (P>0.05), with even smaller Coefficient of Variations (CV) (36.8% for 25% HCA, 35.6% for 25% eugenol) than that from the ICCVAM (66.0% for 25% HCA, 98.8% for 25% eugenol). In addition, the EC1.6 values for HCA, eugenol and DNCB (15.2, 12.5 and 0.25%, respectively) were consistent with that from the ICCVAM (12.92, 8.85 and 0.34%, respectively). DISCUSSION: The results indicate that the reliability for our laboratory to conduct the LLNA:BrdU-ELISA is successfully determined.

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INTRODUCTION: Allergic contact dermatitis (ACD) is a skin disease characterized by eczema and itching. A considerable proportion of chemicals induce ACD in humans. More than 10,000 substances should be tested for skin sensitization potential under the Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH) regulation. The Local Lymph Node Assay (LLNA) has been designated as the first-choice in vivo assay for sensitization testing by REACH. The LLNA:BrdU-ELISA is a validated non-radioactive modification to the LLNA. For both the LLNA and the LLNA:BrdU-ELISA, CBA/JN mouse is the preferred mouse strain recommended in the regulatory guidelines. However, the availability of CBA/JN mouse in China is only limited to a few animal suppliers, which makes the mouse difficult to obtain. BALB/c mouse, which is widely commercially available, is considered for alternative use but it can only be used in the assay after it has been evaluated by formal validation study. Thus, a validation study was conducted in our laboratory to determine if BALB/c mouse could also be used in the LLNA:BrdU-ELISA. METHODS: Forty-three test substances including 32 LLNA sensitizers and 11 LLNA non-sensitizers, their vehicles and each concentration used were the same as that used in the formal validation study for the LLNA:BrdU-ELISA using CBA/JN mouse. Female BALB/c mice of 8-10 weeks old were randomly allocated to groups (four mice per group). The test substance (25 µl) or the vehicle alone was applied to the dorsum of both ears daily for 3 consecutive days. A single intraperitoneal injection of 0.5 ml of BrdU (10mg/ml) solution was given on day 5. On day 6, a pair of auricular lymph nodes from each mouse was excised, weighed and stored at -20°C until BrdU-ELISA was conducted. RESULTS: This validation study for the LLNA:BrdU-ELISA using BALB/c mouse correctly identified 30 of 31 sensitizers and 8 of 11 non-sensitizers. The accuracy, sensitivity, specificity, false positive rate, false negative rate, positive predictivity values and negative predictivity values in this study, which could indicate the performance of the LLNA:BrdU-ELISA using BALB/c mouse, were not different statistically from that of the validation study for the LLNA:BrdU-ELISA using CBA/JN mouse. DISCUSSION: This validation study indicates that BALB/c mouse could be used alternatively in the LLNA:BrdU-ELISA for the prediction of the skin sensitizing potential of chemicals.

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OBJECTIVE: To study the preventive effects of vitamin E on short-term noise-induced hearing loss (NIHL). METHODS: Forty-eight male pigmented guinea pigs were randomly divided into 6 groups, 8 animals in each group. The animals of group 1, 2, 3, 4 were exposed to the noise (4 kHz octave band noise, 100 dB SPL), 8 hours per day for 3 days consecutively and received normal saline, corn oil, 10 mg/kg vitamin E, 50 mg/kg vitamin E respectively daily by intraperitoneal injection from 3 days before the noise exposure, through the 3 noise exposure days to 3 days after the noise exposure. The animals of group 5 and group 6 days were not exposed to the noise but received normal saline and 50 mg/kg vitamin E injection respectively at the same time as that of group 1, 2, 3, 4. The preventive effects of vitamin E on NIHL were determined by comparing the threshold shifts of auditory brainstem responses (ABR) immediately, on the second day and on the 8th day after the exposure. RESULTS: The ABR threshold shifts immediately, on the second day and on the 8th day after the exposure for group 3 at 2, 4 and 8 kHz were (15.9 +/- 6.8), (39.4 +/- 4.8), (42.5 +/- 6.3), (0.3 +/- 2.5), (19.1 +/- 7.9), (21.9 +/- 6.4), (0.3 +/- 1.6), (10.9 +/- 8.6), (12.2 +/- 8.1) dB, respectively, which were significantly lower than those for group 1 [(30.9 +/- 11.3), (47.8 +/- 8.8), (49.7 +/- 6.9), (10.0 +/- 3.5), (29.1 +/- 6.5), (29.1 +/- 7.6), (4.7 +/- 3.6), (20.3 +/- 6.5), (17.5 +/- 9.0) dB, respectively] (P < 0.05). The ABR threshold shifts immediately, on the second day and on the 8th day after the exposure for group 4 at 2, 4 and 8 kHz were respectively (14.4 +/- 5.3), (36.6 +/- 4.4), (43.1 +/- 2.9), (0.3 +/- 2.5), (16.9 +/- 4.6), (19.4 +/- 3.2), (0.0 +/- 3.7), (7.5 +/- 4.2), (9.1 +/- 4.2) dB, which were significantly lower than those for group 1 (P < 0.05). CONCLUSION: Vitamin E has some preventive effects on the NIHL.

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Preventing noise-induced hearing loss (NIHL) by antioxidants is based on the hypothesis that generation of reactive oxygen species is one of the causes of NIHL. alpha-Tocopherol is a naturally occurring antioxidant with no noticeable side effects. In this study, we attempted to protect guinea pigs from developing NIHL by administering alpha-tocopherol. Pigmented male guinea pigs were exposed to a noise (4 kHz octave band, 100 dB SPL), 8 h/day for 3 days consecutively. alpha-Tocopherol (10 mg/kg or 50 mg/kg daily) was given by intraperitoneal injection from 3 days before through 3 days after the noise exposure. Auditory evoked brainstem response (ABR) thresholds at 2, 4 and 8 kHz were recorded prior to the experiment, immediately post-noise, 2 and 8 days post-noise. On day 8 post-noise, after the ABR recording, guinea pigs were decapitated and the cochleae were removed for cochlear surface preparations and scanning electron microscope (SEM) study. ABR threshold shifts of groups receiving alpha-tocopherol were significantly smaller than those of groups not receiving alpha-tocopherol at all frequencies and all time points tested except that of group 3 at 8 kHz 8 days post-noise. No hair cell loss was seen on the surface preparations, but stereocilia loss was found by SEM study. The noise-induced stereocilia loss was significantly decreased by alpha-tocopherol. These results indicate that alpha-tocopherol can attenuate the noise-induced cochlear damage. Further investigations on the preventive effect of alpha-tocopherol on NIHL in noise-exposed workers are necessary.

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OBJECTIVE: To investigate the effect of noise on the antioxidant enzymes of cochleae. METHODS: 16 male pigmented guinea pigs (250 - 300 g) were randomly divided into 2 groups, control group and noise group. Each group had 8 animals. The animals in noise group were performed auditory evoked brainstem responses (ABR) recording before and after exposure to a continuous noise (4 kHz, octave band, 100 dB, SPL) 8 h/d for 3 consecutive days. Immediately at the end of the third day's noise exposure after ABR recording, guinea pigs were decapitated. Both the right and the left cochlea with the bony capsule removed were homogenized, and the supernatants were prepared for assays. Reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured. RESULTS: ROS level of the noise group [(281.2 +/- 3.5) U/mg pro] was significantly higher than that of the control group [(273.0 +/- 3.2) U/mg pro, P < 0.05] and SOD, CAT and GSH-Px activities of the noise group [(206.5 +/- 5.1) NU/mg pro, (47.0 +/- 9.0) U/g pro, (14.1 +/- 2.5) U/mg pro respectively] were significantly lower than that of the control group [(221.8 +/- 4.8) NU/mg pro, (60.8 +/- 9.9) U/g pro, (21.1 +/- 3.1) U/mg pro respectively, P < 0.05]. CONCLUSION: Noise may damage the defensive system of antioxidant enzymes in cochlea.

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