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Toxicological information

Endocrine disrupter mammalian screening – in vivo (level 3)

Administrative data

Endpoint:
endocrine disrupter mammalian screening – in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Benzene Exposure Induces Insulin Resistance in Mice
Author:
Abplanalp WT et al
Year:
2019
Bibliographic source:
TOXICOLOGICAL SCIENCES, 167(2), 2019, 426–437

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Wild type C57BL/6 mice were exposed to volatile benzene (50 ppm x 6 h/day) or HEPA-filtered air for 2 or 6 weeks and measured indices of oxidative stress,
inflammation, and insulin signaling.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzene
EC Number:
200-753-7
EC Name:
Benzene
Cas Number:
71-43-2
Molecular formula:
C6H6
IUPAC Name:
benzene
Test material form:
liquid: volatile
Specific details on test material used for the study:
Benzene atmospheres of either 20 or 50 ppm were generated from liquid benzene (Sigma-Aldrich) in a KIN-TEK Analytical, Inc permeation tube. A carrier gas (N2) was delivered to the permeation tube at 100 ml/min and diluted with HEPA- and charcoalfiltered room air (HFA; 3 L/min) and diluted gas directed to an exposure unit.

Test animals

Species:
mouse
Strain:
C57BL
Details on species / strain selection:
Ten-week old male C57BL/6 mice
Sex:
male
State:
not specified
Details on test animals and environmental conditions:
Ten-week old male C57BL/6 mice were obtained from Jackson Laboratories and were provided normal chow and water ad libitum throughout exposures. In some experiments, mice were placed on drinking water containing 1 mM TEMPOL (4-Hydroxy TEMPO: Sigma-Aldrich) starting 3 days prior to initiation of exposure and continuing throughout the exposure duration. Drinking water was changed daily.

Administration / exposure

Route of administration:
other: inhalation
Details on route of administration:
Benzene atmospheres were generated from liquid benzene (Sigma-Aldrich) in a KIN-TEK Analytical, Inc permeation tube. A carrier gas (N2) was delivered to the permeation tube at 100 ml/min and diluted with HEPA- and charcoalfiltered room air (HFA; 3 L/min) and diluted gas directed to an
exposure unit. Flow was distributed through a fine mesh screen. of a custom cyclone-type top (Teague Enterprises) that distributed air within 10% of the mean concentration at six locations in the cage. Throughout an exposure, benzene concentrations were continuously monitored using an in-line photoionization detector (ppb RAE: Rae Industries) upstream of the exposure unit. Mice were exposed to 50 ppm benzene for 6 h/day for 2 or 6 weeks or to 10 ppm benzene for 6 h/day for 2 weeks. Mice exposed to HFA only were used as a control. Exposures in individual animals were assessed by measuring the urinary levels of the benzene metabolite, muconic acid, t,t-MA. All procedures were approved by the University of Louisville Institutional Animal Care and Use Committee.
Vehicle:
other: Nitrogen carrier gas
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Throughout an exposure, benzene concentrations were continuously monitored using an in-line photoionization detector (ppb RAE: Rae Industries) upstream of the exposure unit.
Exposures in individual animals were assessed by measuring the urinary levels of the benzene metabolite, muconic acid, t,t-MA
Duration of treatment / exposure:
6 hours/day for 2 or 6 weeks for 50ppm exposure
6 hours/day for 2 weeks for 10ppm exposure
Mice exposed to HFA only were used as a control.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 ppm
Dose / conc.:
50 ppm
Control animals:
yes, concurrent vehicle
Details on study design:
Mice exposed to HFA only were used as a control.

Systemic effects, effects on glucose handling, and oxidative stress/inflammation were analysed by exposure to 50ppm of volatile benzene (HFA for control) for 6h/day 2 weeks initially, additional exposures for 6 weeks.

Examinations

Observations and examinations performed and frequency:
Analysis of urinary metabolites.
Urine was collected in chilled tubes from mice housed in metabolic cages for 18 h. The urine was then centrifuged at 400 x g for 5 min and the supernatant stored at -20°C. Aliquots of these samples (20 ml) were mixed with an equal volume of 0.5M sodiumphosphate, pH 8.0 containing 10 mM t,t-MA as an internal standard and incubated with 130 ml 0.1 M PFBBr for 60 min at 50C, and then extracted with 300 ml hexane. The extract (100 ml) was removed and analyzed on an Agilent 6890 N GC-MS with chemical ionization ion source. Sixpoint calibration curves were used to calculate the concentrations of t,t-MA (2.5 mM to 100 mM). Levels of urinary t,t-MA were normalized to levels of creatinine, which were measured on a COBAS MIRA-plus analyzer with Infinity Creatinine Reagent (Thermo Fisher Scientific,Massachusetts) (Srivastava et al., 2011).

In vivo assessment of glucose handling.
Plasma glucose was measured following a 6 h fast using a standard glucose meter (Accucheck, Aviva) and glucose test strips (Accu-check, Aviva Plus). Fasting plasma insulin was measured by ELISA (Mercodia). HOMA-IR was calculated using the equation: HOMA-IR ¼ (FPG [mg/dl]*FPI [mIU/l])/405, whereas HOMA-b was determined using the equation: HOMA-b ¼ (20*FPI [mIU/l])/(FPG [mmol/l]-3.5) (Matthews et al., 1985).

For glucose tolerance tests, (GTT) mice were fasted for 6 h and D-glucose (1 mg/g body weight, i.p.) was given as previously described in McGuinness et al. (2009). Insulin tolerance tests (ITT) were performed on unfasted animals by injection of Humulin R (Eli Lilly; 1.0 U/kg body weight).
Sacrifice and pathology:
Complete blood cell counts and plasma biochemistry.
At end of the exposure protocol, mice were euthanized with sodium pentobarbital (150 mg/kg body weight; i.p. 100 ll of solution in PBS). Peripheral blood was collected in 0.2 M EDTA-coated syringes by cardiac puncture and an aliquot of 25 ml was used for complete blood count on a Hemavet 950FS (Coulter). Plasma levels of total protein, albumin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured on a COBAS MIRAplus analyzer (Roche, New Jersey). For glucose tolerance tests, Areas under the curve (AUC; GTT) and area above the curve (AAC; ITT) were calculated using the trapezoid rule with subtraction of baseline glucose area (Conklin et al., 2017).

Western blotting and qPCR

Liver and skeletal muscle homogenates were prepared in chilled RIPA buffer using a Dounce homogenizer and then centrifuged at 4°C for 10 min at 17 000 x g. The supernatants were collected and protein concentrations were measured using a Bradford assay. For analysis of Nfjb p65 and phospho-Nfjb p65, nuclear extracts were prepared using the Episeeker Nuclear Extraction kit (Abcam). Western blotting with specific antibodies (Supplementary Table 1) was performed to analyze the levels of Akt, phospho-Akt Ser473, Nfjb p65, phospho-Nfjb p65 Ser536, Socs1, and Socs3. Images were acquired using a Typhoon 7000 FLA imaging system (GE Healthcare) and band intensities were quantified using the ImageJ software (NIH.gov). For quantitative rtPCR, RNA was extracted from frozen tissues using the miRNEasy isolation kit (Qiagen). Levels of Mip-1a, IL-1b, IL-6, and Tnfa were then measured using specific primers (Integrated DNA Technologies; Supplementary Table 2) and the Universal SYBR Green PCR Master Mix (Stratagene) on an Applied Biosystems 7900HT Fast Real Time instrument. GAPDH was used as an internal control.

Immunohistochemistry

Formalin-fixed, paraffin-embedded liver sections (4 mm) were de-paraffinized and rehydrated by sequential immersion in a graded series of alcohol and water. The sections were heated in amicrowave oven for 10min in 10mM sodium citrate buffer for epitope retrieval. After washing with PBS, pH 7.4, sections were blocked with 10% fetal calf serum for 30 min and then incubated with an anti-CD68 antibody (Abcam) at a 1:20 dilution for 18 h at 4°C, followed by incubation with TRITCconjugated goat anti-rabbit IgG for 60 min at room temperature. Stained sections were visualized on a Nikon Eclipse Ti fluorescent microscope using a 20x objective and images were acquired.

Because benzene exposure perturbs glucose homeostasis and induces a state of systemic glucose intolerance, we examined whether exposure to benzene interfered with insulin signaling.

Measurement of oxidatice stress

Glutathione (GSH) levels were measured in frozen liver and skeletal muscle samples using the BIOXYTECH GSH-412TM Colorimetric Determination Glutathione Kit (Oxis Research). To measure intracellular GSH in leukocytes, cell preparations were incubated with monochlorobimane (40 lM) for 20 min at room temperature and immunofluorescence was analyzed on an LSRII flow cytometer (Becton Dickinson). To measure the lipid peroxidation product malondialdehyde (MDA), a Lipid Peroxidation (MDA) Assay Kit (Sigma) was used.
Statistics:
Data are presented as mean 6 standard error of the mean. All data were analyzed using GraphPad Prism version 5.0 software (GraphPad Software, La Jolla, California). Data from inhalation exposure experiments are derived from multiple exposures. The data were analyzed and compared by two-tailed Student’s t tests, except where comparisons were made across more than two groupings using one-way ANOVA as appropriate. A p value of < .05 was considered statistically significant.

Results and discussion

Endocrine disrupting potential:
not specified
Maximum tolerated dose level exceeded:
yes

Results of examinations

Description (incidence and severity):
Systemic effects of benzene exposure:
After 2 weeks of exposure, benzene only affected neutrophil and red blood cell counts (Supplementary Table 3). However, mice that were exposed to benzene for 6 weeks had significantly lower levels of circulating white blood cells, neutrophils, lymphocytes, monocytes, and platelets than mice breathing filtered air.
Benzene-exposed mice also demonstrated significantly elevated plasma ALT and AST levels compared with mice breathing filtered air. In comparison with control mice, mice exposed to benzene for 6 weeks had higher levels of plasma albumin. These observations suggest that exposure to 50 ppm benzene not only affects hematology but also induces mild liver injury in mice

Description (incidence and severity):
Systemic effects of benzene exposure:
Two weeks of exposure to benzene also increased the liver:body weight ratio (Supplementary Table 3), but had no effect on overall body weight or growth.

Effect levels

Dose descriptor:
LOEL
Effect level:
50 other: ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Clinical chemistry (glucose intolerance)

Any other information on results incl. tables

Glucose handling

Given the marked systemic effects of benzene, we next examined its effects on glucose handling. We found that mice exposed to 50 ppm benzene for 2 weeks had a 1.13-fold higher level of fasting plasma glucose and a 1.39-fold higher level of fasting plasma insulin than mice breathing filtered air, suggesting that exposure to benzene might induce insulin resistance. To quantify this effect, we calculated HOMAIR scores, that were significantly higher in benzene-exposed mice than control mice. As with HOMA-IR, the values of HOMA-b were also significantly higher in benzene-exposed mice. Mice exposed to filtered air or a lower dose of benzene (10 ppm) for 2 weeks demonstrated no differences in fasting plasma glucose, fasting plasma insulin, HOMA-IR scores and HOMA-b scores. Mice exposed to 50 ppm benzene for a longer duration (6 weeks) had stronger, statistically significant differences versus their air counterparts in both insulin levels (p ¼ .0001 vs p ¼ .03) and HOMA-IR scores (p ¼ .001 vs 0.005). Similarly, GTT in mice exposed to 50 ppm benzene, but not 10 ppm benzene had a modest, but significant increase in the GTT AUC relative to control mice. Congruently, the ITT and AAC calculations showed an appreciable decrement in insulin responsiveness in mice inhaling 50 ppm benzene, indicating that more insulin was required to sequester glucose than in control mice. Given that 10 ppm benzene had no effect on glucose handling and that a 2 weeks of exposure at 50 ppm was effective, all further experiments were done using this level and exposure duration.

Because benzene exposure perturbs glucose homeostasis and induces a state of systemic glucose intolerance, we examined whether exposure to benzene interfered with insulin signaling. For this, we injected exposed animals with insulin 15 min before euthanasia and then analyzed Akt phosphorylation in insulin-sensitive organs. We found a marked increase in Akt phosphorylation in the liver of air-exposed mice (2.82-fold induction), but an attenuated increase in mice exposed to benzene (1.02-fold increase). Similar changes were observed in skeletal muscle, which also showed significant deficits in insulin-induced Akt phosphorylation in benzeneexposed mice. Collectively, these observations suggest that exposure to benzene induces organ level insulin resistance that likely contributed to systemic glucose and insulin intolerance.

Oxidative stress and inflammation

Levels of GSH in liver homogenates decreased from 12.2 6 0.7 mmoles/g tissue in air-exposed mice to 9.0 6 1.3 mmoles/g tissue in benzene-exposed mice. A similar decrease in GSH levels was observed in skeletal muscle. To examine the effects of benzene on GSH levels in blood leukocytes, we loaded these cells with monochlorobimane (MCB), a fluorescent GSH-binding dye, and measured changes in fluorescence by flow cytometry. Median MCB fluorescence (ie, GSH levels) was significantly higher in leukocytes from air-exposed mice than from benzene-exposed mice (259 6 31 vs 89 6 15 A.U.). Taken together, these results show that in several organs and blood, benzene exposure depleted GSH, indicating systemic oxidative stress. The notion that benzene induces oxidative stress was further corroborated by the observation that hepatic levels of malondialdehyde (MDA), a lipid peroxidation product, were higher in the benzene-exposed animals relative to controls (0.41 6 0.01 vs 0.24 6 0.01 nmol/mg protein).

As oxidative stress can trigger inflammation, we next examined markers of inflammation by assessing changes in the phosphorylation of the p65 subunit of Nfjb. We found in both the liver and skeletal muscle that phosphorylation of p65 was increased following exposure to benzene. To further support the idea that benzene exposure caused liver inflammation, we examined the presence of monocytes/macrophages in that tissue by immunohistochemistry. Whereas liver sections obtained from mice breathing HFA showed little positive staining with an anti-CD68 antibody those sections from mice inhaling

benzene showed stronger staining with this antibody.

As phosphorylation of p65 results in increased expression of several cytokines, we measured transcript levels of a panel of cytokines by quantitative rtPCR. Whereas hepatic levels of IL-1b and Tnfa were unaffected after 2 weeks of exposure, there was a significant (1.94-fold) increase in levels of Mip-1a in benzeneexposed mice relative to air-exposed mice. Benzeneexposure also increased Mip-1a in skeletal muscle. Because Mip-1a increases transcription of Socs1 and Socs3, which are known to attenuate insulin signaling through inhibition of Irs phosphorylation (Qin et al., 2008), we next measured hepatic levels of these proteins. These measurements showed that there was a significant increase in Socs1 (1.74-fold) in benzene-exposed mice and a trending increase in Socs3 (1.28-fold, p ¼ .058). Finally, because Socs1 can inhibit Irs-2 phosphorylation, we examined the phosphorylation of this protein. We found a significant decrease in insulinstimulated tyrosine phosphorylation of Irs-2 in the liver of benzene-exposed mice. Collectively, these observations show that benzene-induced oxidative stress and inflammation may limit insulin responsiveness (Irs-2 phosphorylation) through up-regulation of Socs1.

TEMPOL Attenuated the Effects of Benzene

To determine if a reduction of oxidative stress would mitigate the effects of benzene, we placed mice on drinking water containing the anti-oxidant TEMPOL (4-hydroxy TEMPO). To examine the efficacy of this treatment, we first measured organ GSH levels. We found that TEMPOL attenuated GSH depletion in the liver of benzene-exposed mice. GSH-depletion in skeletal muscle was also attenuated by TEMPOL. To measure oxidative stress more directly, we quantified MDA levels. Benzene-exposed mice receiving TEMPOL-containing drinking water had significantly lower concentrations of hepatic MDA than benzene-exposed animals drinking normal water (0.19 vs 0.41 nmole/mg protein). Treatment with TEMPOL also mitigated inflammation as evidenced by decreased levels of phosphorylated Nfjb p65 in both the liver and skeletal muscle. Likewise, levels of Mip-1a transcripts in liver and skeletal muscle were decreased in the benzene-exposed animals drinking TEMPOL-containing water. Taken together, these observations suggest that inflammation in benzene-exposed mice was likely attributable to oxidative stress.

Because TEMPOL limited benzene-induced inflammation, we next determined if this treatment would also limit the effects of benzene on insulin signaling. First, we observed that benzene-exposed mice receiving TEMPOL had decreased expression of Socs1 in the liver compared with mice drinking normal water. Second, corresponding to decreased Socs1 expression, we found that benzene-exposed, TEMPOLtreated animals exhibited increased insulin-stimulated Irs-2 tyrosine phosphorylation relative to benzene-exposed mice drinking normal water. Third, we observed that benzene-exposed, TEMPOL-treated animals maintained their responsiveness to insulin and had higher levels of Akt phosphorylation in both the liver and skeletal muscle than benzene-treated mice drinking normal water.

Finally, we determined whether treatment with TEMPOL would prevent the effects of benzene on glucose handling. We found that benzene-exposed mice receiving TEMPOL had significantly lower levels of fasting plasma glucose and fasting plasma insulin than did their benzeneexposed counterparts drinking normal water. TEMPOL treatment also lowered HOMA-IR scores in benzene-treated mice (Figure 8C), and these mice displayed glucose tolerant responses to either a glucose or an insulin bolus in GTT and ITT assays, respectively. These observations suggest that benzene-induced insulin resistance is mediated by oxidative stress.

Applicant's summary and conclusion

Conclusions:
The data show that exposure to volatile benzene can induce metabolic disorders. These findings are consistent with previous work showing that benzene exposure in humans was associated with increased cardiovascular disease risk scores and deficits of vascular reparative cells (Abplanalp et al., 2017). This study raises the possibility that occupational exposure to benzene may be associated with heightened risk for the development of metabolic disease in exposed workers. Therefore, increased workplace surveillance is required to minimize such exposures. Finally, given that benzene is ubiquitous in urban environments and is generated in high concentrations in automobile exhaust, it is tempting to speculate the current high prevalence of insulin resistance and diabetes in urban areas may be in part linked to benzene exposure and that minimizing benzene emissions may be a readily implementable strategy to assist in decreasing the global burden of insulin resistance and diabetes.
Executive summary:

Benzene is a ubiquitous pollutant associated with hematotoxicity but its metabolic effects are unknown. We sought to determine if and how exposure to volatile benzene impacted glucose handling. We exposed wild type C57BL/6 mice to volatile benzene (50 ppm x 6 h/day) or HEPA-filtered air for 2 or 6 weeks and measured indices of oxidative stress, inflammation, and insulin signaling. Compared with air controls, we found that mice inhaling benzene demonstrated increased plasma glucose (p ¼ .05), insulin (p ¼ .03), and HOMA-IR (p ¼ .05), establishing a state of insulin and glucose intolerance. Moreover, insulin-stimulated Akt phosphorylation was diminished in the liver (p ¼ .001) and skeletal muscle (p ¼ .001) of benzene-exposed mice, accompanied by increases in oxidative stress and Nf-jb phosphorylation (p ¼ .025).

Benzene-exposed mice also demonstrated elevated levels of Mip1-a transcripts and Socs1 (p ¼ .001), but lower levels of Irs-2 tyrosine phosphorylation (p ¼ .0001). Treatment with the superoxide dismutase mimetic, TEMPOL, reversed benzeneinduced effects on oxidative stress, Nf-jb phosphorylation, Socs1 expression, Irs-2 tyrosine phosphorylation, and systemic glucose intolerance. These findings suggest that exposure to benzene induces insulin resistance and that this may be a sensitive indicator of inhaled benzene toxicity. Persistent ambient benzene exposure may be a heretofore unrecognized contributor to the global human epidemics of diabetes and cardiovascular disease.