In Utero Maternal Benzene Exposure Predisposes to the Metabolic Imbalance in the Offspring

Abstract

Environmental chemicals play a significant role in the development of metabolic disorders, especially when exposure occurs early in life. We have recently demonstrated that benzene exposure, at concentrations relevant to cigarette smoke, induces a severe metabolic imbalance in a sex-specific manner affecting male but not female mice. However, the roles of benzene in the development of aberrant metabolic outcomes following gestational exposure, remain largely unexplored. In this study, we exposed pregnant C57BL/6JB dams to benzene at 50 ppm or filtered air for 6 h/day from gestational day 0.5 (GD0.5) through GD21 and studied male and female offspring metabolic phenotypes in their adult life. While no changes in body weight or body composition were observed between groups, 4-month-old male and female offspring exhibited reduced parameters of energy homeostasis (VO₂, VCO_2, and heat production). However, only male offspring from benzene-exposed dams were glucose intolerant and insulin resistant at this age. By 6 months of age, both male and female offspring exhibited marked glucose intolerance however, only male offspring developed severe insulin resistance. This effect was accompanied by elevated insulin secretion and increased beta-cell mass only in male offspring. In support, Homeostatic Model Assessment for Insulin Resistance, the index of insulin resistance was elevated only in male but not in female offspring. Regardless, both male and female offspring exhibited a considerable increase in hepatic gene expression associated with inflammation and endoplasmic reticulum stress. Thus, gestational benzene exposure can predispose offspring to increased susceptibility to the metabolic imbalance in adulthood with differential sensitivity between sexes.

adverse health outcomes, gestational exposure, offspring diabetes, glucose metabolism, benzene exposure, energy homeostasis, air pollution, developmental toxicity, endocrine disruptors, inflammation

Type 2 diabetes (T2DM) is an escalating public health concern with complex genetic, behavioral, and environmental origins (Brancati et al., 1996; Fujimoto, 1996; Shaw et al., 2010). T2DM mostly results from the interaction between genetic, environmental, and other risk factors (Murea et al., 2012). Emerging evidence suggests that environmental chemical exposures may contribute to T2DM by altering whole-body glucose metabolism, insulin resistance, as well as by affecting satiety signals and energy homeostasis in the central nervous system (Kuo et al., 2013). Compelling evidence indicates that exposure to various toxins or air pollution during sensitive periods of early development causes a predisposition to metabolic disorder later in life (Das et al., 2018; Rauh and Margolis, 2016; Soh et al., 2018; Suryadhi et al., 2019; Vanker et al., 2018). Specifically, volatile organic compounds (VOCs) have been shown to induce damaging health effects (Dezest et al., 2017; Mohamed et al., 2002; Williams et al., 2019; Yoon et al., 2010).

Benzene is one of the main VOCs found in ambient air pollution (Kliucininkas et al., 2011). Other sources of benzene include processed foods, occupational exposure, vehicle and manufacturing emissions, cigarette smoke, and electronic cigarette fumes (Duarte-Davidson et al., 2001; Minciullo et al., 2014; Pankow et al., 2017; Salviano Dos Santos et al., 2015; Wallace, 1989; Williams and Mani, 2015). Benzene is known to be carcinogenic at high concentrations, but its association in causing metabolic diseases particularly T2DM is still emerging. Recent studies demonstrated that benzene’s noncancerous health effects at the levels relevant to smoking or those present in vehicle emissions include impaired glucose homeostasis and insulin resistance (Abplanalp et al., 2017; Amin et al., 2018). Early studies estimated that in each puff of tobacco products there is approximately 20–100 ppm benzene (Appel et al., 1990). The 1999 Massachusetts Benchmark Study reported an average benzene delivery of 86 μg/cigarette (Harris, 2004), which based on the proposed protocol corresponds to a smoke benzene concentration of approximately 62 ppm (Pankow et al., 2017). We have recently shown that chronic exposure of adult mice to benzene at 50 ppm induces severe metabolic imbalance associated with central hypothalamic inflammation and endoplasmic reticulum (ER) stress in a sex-specific manner (Debarba et al., 2020). These data indicate that exposure to benzene can affect an individual’s susceptibility to metabolic imbalance and the development of T2DM.

Most epidemiological studies investigating the association between benzene exposure and insulin resistance have focused on young adult or elderly populations living in highly polluted areas (Abplanalp et al., 2017; Burg and Gist, 1998; Choi et al., 2014). However, very few studies have assessed the risk of benzene exposure during pregnancy on the long-term metabolic health of the grown offspring. Recent epidemiological studies have highlighted the association of benzene exposure with increased risk for gestational diabetes (Williams et al., 2019). Similarly, epidemiological data have shown a positive association between cigarette smoke exposure in utero and increased risk of obesity and metabolic imbalance in offspring (Behl et al., 2013). A study of traffic pollutants (PM10 and benzene) in a community in northern Italy showed an association between exposure to PM10 and different birth defects (De Donno et al., 2018). However, the direct link between maternal exposure to benzene during pregnancy and metabolic disease in offspring is not clear.

This study sought to determine whether exposure of pregnant females to benzene alters the susceptibility of the young and/or adult offspring to metabolic imbalance. Pregnant females were exposed to filtered air or concentrations of benzene at the levels relevant to cigarette smoke throughout the gestation by direct inhalation for 6 h per day, 5 days a week. Under these conditions, benzene exposure had no significant effect on litter size or composition and no significant changes in animals’ white blood cell counts (Debarba et al., 2020). Our findings indicate that maternal exposure to benzene induces deleterious effects on energy expenditure, glucose metabolism, and insulin resistance in adult offspring in a sex-specific manner, providing evidence that gestational benzene exposure is a potential risk factor for metabolic disorders.

MATERIALS AND METHODS

Animals

Eight- to nine-week-old male and female C57BL/6 were purchased from The Jackson Laboratory. After a 1-week habituation period, female and male mice were mated, and mice were considered pregnant if a vaginal plug was observed. Upon discovery of a vaginal plug (gestational day 0.5 [GD0.5]), females were removed, weighed, and randomly assigned to a treatment group. Pregnant dams were exposed to benzene at 50 ppm or filtered air using a whole-body exposure chamber (as described below) for 6 h/day from GD0.5 through GD21 (5–6 pregnant dams per group). Exposures occurred between 09:00 and 15:00 h during the light cycle. Dams had free access to food and water ad libitum. At birth and during the postnatal period dams were maintained in ambient air. No offspring mortality was observed at birth or postnatally. Final offspring sample numbers were: 6–7 males/group control or benzene offspring and 5–6 females/group control or benzene offspring from 5 to 6 litters/treatment group. All pups remained with their dams until P21, at which time they were group-housed by litter and sex and continued on the control chow diet (Purina Lab Diet 5001). Most of the presented data related to both male and female mice unless otherwise stated.

Benzene exposure

Pregnant dams in inhalation chambers using FlexStream automated Perm Tube System (KIN-TEK Analytical, Inc) were exposed to benzene concentration of 50 ppm (Debarba et al., 2020). FlexStream automated Perm Tube System allows creating precision gas mixtures. This unit provides a temperature-controlled permeation tube oven, dilution flow controls, and front panel touch-screen interface. Mixtures are produced by diluting the miniscule flow emitted from Trace Source permeation tubes with a much larger flow of inert matrix gas, typically nitrogen or zero air. Control dams were breathing filtered air. All mice were provided with water ad libitum and housed in temperature-controlled rooms on a 12/12-h light-dark cycle.

Metabolic analysis

Lean and fat body mass were assessed by a Bruker Minispec LF 90II NMR-based device. Intraperitoneal glucose tolerance test (GTT) was performed on mice fasted for 5–6 h. Animals were then injected intraperitoneally with D-glucose (2 g/kg) and blood glucose levels were measured as before (Sadagurski et al., 2010). For an insulin tolerance test (ITT), animals fasted for 4 h received an intraperitoneal injection of insulin (Humulin R, 0.8 U/kg) diluted in sterile saline. Blood glucose concentrations were measured at the indicated time points. Blood insulin levels were determined on serum from tail vein bleed using a Mouse Insulin ELISA kit (Crystal Chem. Inc.). In all tests, tail blood glucose levels were measured at the indicated times after injection. Metabolic measurements of energy homeostasis were obtained using an indirect calorimetry system (PhenoMaster, TSE system, Bad Homburg, Germany). The mice were acclimatized to the cages for 2 days and monitored for 5 days while food and water were provided ad libitum.

Histology and morphometric analysis

Histological analysis for insulin-stained pancreatic β-cell tissues was performed as previously described (Sadagurski et al., 2014). Briefly, pancreas sections were rehydrated using xylene and decreasing concentrations of EtOH, permeabilized using 0.2% Triton X-100/PBS, and antigen unmasking was performed in a boiling solution of 0.01 M sodium citrate (pH 6.0) for 20 min. Sections were blocked with normal goat serum for 1 h and incubated overnight with primary guinea pig anti-insulin (1:100; Zymed Laboratories, San Francisco, California) antibody. Alexa-Flour (Molecular Probes, Eugene, Oregon) secondary antibody was used (1:200). The β-cell mass (mg per pancreas) was calculated by point-counting morphometry from insulin-stained sections (5 μm) separated by 200 μm using the BQ Classic98 MR software package (BIOQUANT) and corrected to the total area of the pancreas section as described (Elghazi et al., 2007; Sadagurski et al., 2014).

RNA extraction and qPCR

Total RNA was extracted from the liver with Trizol (Gibco BRL) and 1000 ng of RNA was used for cDNA synthesis using iscript cDNA kit (Bio-Rad Laboratories Inc.). Quantitative real-time PCR was performed using the Applied Biosystems 7500 Real-Time PCR System. The following SYBER Green Gene Expression Assays (Applied Biosystems) were used in this study. (See Supplementary Table 1 for primers sequence.) Each PCR reaction was performed in triplicate. Lack of reverse transcription was used as a negative control for contaminating DNA detection. Water instead of cDNA was used as a negative control, and the housekeeping gene ß-actin was measured in each cDNA sample. Gene transcripts in each sample were determined by the ΔΔCT method. For each sample, the threshold cycle (CT) was measured and normalized to the average of the housekeeping gene (ΔCT). The fold change of mRNA in the unknown sample relative to the control group was determined by 2^−ΔΔCT. Data are shown as mRNA expression levels relative to the control group.

Statistical analysis

Data sets were analyzed for statistical significance using Statistica software (version 10). An analysis of variance with repeated measurements was used to analyze body weight, GTT, and ITT tests. Additionally, an analysis for a 2-tail unpaired Student’s t test was used when 2 groups were compared. The level of significance (α) was set at 5%.

RESULTS

Gestational Benzene Exposure Does Not Alter Body Weight in the Offspring

We exposed pregnant females to benzene at the concentration of 50 ppm throughout the pregnancy using an exposure chamber for 6 h/day, from the day the vaginal plug was detected to birth. Figure 1A illustrates a schematic timeline of the experimental design. This adapted regimen for benzene inhalation resembles chronic human benzene exposure levels in highly polluted gas stations, or exposure to tobacco smoke (De Donno et al., 2018; Lorkiewicz et al., 2019; Pankow et al., 2017). We have recently demonstrated that under these conditions, benzene exposure has no significant changes in white blood cell counts in benzene-exposed animals versus control animals breathing room air (Debarba et al., 2020). Maternal exposure to benzene did not significantly affect male and female offspring body mass at weaning and throughout adulthood (Figs. 1B and 1C). Additionally, the lean and fat body mass of male and female offspring were not affected by maternal benzene exposure (Figs. 1D and 1E).

Figure 1.

Gestational benzene exposure does not alter body weight in the offspring. A, Experimental design timeline. Effect of gestational benzene exposure on body weight gain (g) of males (B) and females (C); fat mass (g), lean mass (g) and total mass (g) of males (D) and females (E) measured by magnetic resonance imaging (echo MRI), data are expressed as the mean ± SEM (n = 6). Repeated measures analysis of variance were further analyzed with Newman-Keuls post hoc analysis or t test if necessary to compare between only two groups of the predictor variable.

Gestational Benzene Exposure Predisposes to Impaired Energy Expenditure and Glucose Metabolism in Offspring

We next assessed whole-body energy homeostasis in young adult male and female offspring (Figs. 2 and 3). Both male and female offspring of benzene-exposed dams demonstrated significantly decreased oxygen consumption (VO₂), carbon dioxide (VCO₂) production, and heat production during the 24-h period (p < .01, Figs. 2A–C and 3A–C). Interestingly, male offspring of benzene-exposed dams exhibited a greater reduction in energy expenditure parameters compared with female offspring as indicated by stronger reduction in VO₂, VCO₂ and heat production during both light and dark cycles (p < .01, Figs. 2A–C and 3A–C). There was no significant difference in respiratory exchange ratio, locomotor activity levels, or food intake among the groups (Figs. 2D and 3D, Supplementary Figure 1, and data not shown).

Figure 2.

Gestational benzene exposure impairs energy homeostasis in 4-month-old male offspring. Effect of gestational benzene on (A) oxygen consumption (VO₂—ml/kg/h); (B) carbon dioxide production (VCO₂—ml/kg/h); (C) heat production (kcal/h/kg); (D) locomotor activity during light, dark, and the entire 24 h cycles of male offspring at 4 months of age. The line graphs depict variations throughout each cycle. Data are expressed as the mean ± SEM (n = 5–6/group). Repeated measures analysis of variance were further analyzed with Newman-Keuls post hoc analysis or t test if necessary, to compare between only 2 groups of the predictor variable (* = vs control; *p < .05; **p < .01).

Figure 3.

Gestational benzene exposure impairs energy homeostasis in 4-month-old female offspring. Effect of gestational benzene on (A) oxygen consumption (VO₂—ml/kg/h); (B) carbon dioxide production (VCO₂—ml/kg/h); (C) heat production (kcal/h/kg); (D) locomotor activity during light, dark, and the entire 24 h cycles of male offspring at 4 months of age. The line graphs depict variations throughout each cycle. Data are expressed as the mean ± SEM (n = 5–6/group). Repeated measures analysis of variance were further analyzed with Newman-Keuls post hoc analysis or t-test if necessary, to compare between only 2 groups of the predictor variable (* = vs control; *p < .05; **p < .01).

Exposure of male mice to benzene during gestation resulted in severe hyperglycemia, as indicated by GTT (Figure 4A), and by the significantly higher area under the curve and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), an index of insulin resistance (Figs. 4A, 4C, and 4D) in 4-month-old male offspring. On the other hand exposure of female mice to benzene during gestation had no effect on glycemia at this age (Figure 4B). However, by 6-month of age both male and female offspring of benzene-exposed dams exhibited marked glucose intolerance (Figs. 5A and 5B). By this age male offspring of benzene-exposed dams developed severe insulin resistance (p < .05), whereas female offspring, demonstrated a slight impairment in insulin tolerance (p < .05, Figs. 5C and 5D). To examine glucose-stimulated insulin secretion in 6-month-old male offspring, we measured plasma insulin at baseline, 15 min, and 30 min after i.p. glucose challenge (Figure 6A). Although insulin fasting levels were not different between the groups, the overall glucose-stimulated insulin secretory response was significantly increased in the male offspring of benzene-exposed dams (p < .05 vs control offspring). Additionally, maternal benzene exposure modulated islet morphology in these male offspring and they were characterized by a significant increase in β-cells mass compared with control male offspring (Figs. 6B and 6C). Notably, β-cell mass in female offspring of benzene-exposed dams was similar to control female offspring (Supplementary Figure 2), confirming the observed sex differences in metabolic adaptation to benzene exposure (Debarba et al., 2020). In support, of the observed metabolic impairment, HOMA-IR was significantly elevated in male but not in female offspring of benzene-exposed dams compared with control offspring at 9 months of age (p < .05, Figs. 7A and 7B).

Figure 4.

Gestational benzene exposure impairs glucose metabolism in the offspring in a sex-specific manner at 4 months of age. Effect of gestational benzene on glucose tolerance test of (A) male and (B) female offspring; (C) Area under the curve; (D) HOMA-IR of male offspring, at 4 months of age. Data are expressed as the mean ± SEM (n = 6–7/group). Repeated measures analysis of variance were further analyzed with Newman-Keuls post hoc analysis or by t test if necessary to compare between only 2 groups of the predictor variable (*p < .05 vs control).

Figure 5.

Gestational benzene exposure impairs glucose and insulin tolerance in the offspring at 6 months of age. Effect of gestational benzene on glucose tolerance test of (A) male and (B) female offspring; Insulin tolerance test of (C) male and (D) female offspring. Data are expressed as the mean ± SEM (n = 6–7/group). Repeated measures analysis of variance were further analyzed with Newman-Keuls post hoc analysis (*p < .05 vs control).

Figure 6.

Gestational benzene exposure impairs insulin secretion and beta cells mass in male offspring. A, Insulin secretion. B, Immunofluorescence staining for insulin (green) and 4',6-Diamidino-2-Phenylindole (blue) in representative pancreatic sections from 9-month-old male offspring. C, Quantitation of β-cell mass in male offspring. Data are expressed as the mean ± SEM (n = 6–7/group; *p < .05 vs control).

Figure 7.

Gestational benzene exposure alters the hepatic gene expression of offspring. HOMA-IR of (A) male offspring and (B) female offspring at 9 months of age. qPCR of hepatic inflammatory and ER stress genes (Ikbkb, Ikbke, TNFα, Il6, and Chop) of (C) males and (E) females. qPCR of hepatic gluconeogenesis genes (Pepck and Gck) of (D) males and (F) females. Data were expressed as the mean ± SEM (n = 4–5) and analyzed by t test (* = vs control; *p < .05, **p < .01; ***p < .001).

To further investigate the potential mechanisms of metabolic imbalance in benzene-exposed offspring, we analyzed the mRNA levels of genes associated with gluconeogenesis, lipid metabolism, and inflammation in the livers of male and female offspring (Figs. 7C–F). The inflammatory signature was observed in both 9-month-old male and female offspring of benzene-exposed dams; however, the effect was more striking in the male offspring, with a significant increase in hepatic inflammatory genes Ikke, Ikkb, TNFα, and Il6 (Figs. 7C and 7D). Both male and female offspring exhibited an increase of—CCAAT/enhancer binding protein homologous protein (Chop), a gene involved with the ER-stress pathway (Figs. 7C and 7E). We also detected an increase in the expression of genes associated with gluconeogenesis (Pepck and Gck) in livers from benzene-exposed male offspring as compared with control offspring mice (Figure 7D). Gck was similarly elevated in livers from benzene-exposed female offspring, without significant differences in Pepck expression (Figure 7F). No differences were observed in some additional genes associated with inflammation and ER stress, such as Emr1, Ire1a, and Xbp1s, or genes associated with lipid and fatty acids synthesis (Supplementary Figure 3). Additionally, we did not detect any differences in the expression of genes for insulin signaling (such as Irs1 and Foxo1) in the muscle from benzene-exposed offspring, while the expression of the glucose transporter Glut1 was significantly decreased in the muscle of male but not female offspring (Supplementary Figs. 4A and 4B). No difference in the expression of Irs1, Foxo1, or in the expression of lipid metabolic gene, Srebp1 was observed between groups in the adipose tissue (Supplementary Figs. 4C and 4D).

To address the potential predisposition to inflammatory phenotype and metabolic imbalance early in development we have exposed pregnant females to 2 concentrations of benzene (50 ppm or to a lower dose of 5 ppm) for 6 h/day from GD0.5 to GD17.5 in the inhalation chambers and analyzed the expression of inflammatory genes and gluconeogenesis genes in the liver of the fetuses at GD17.5. As can be seen in Supplementary Figure 5, there is an increase in the expression of inflammatory genes (TNFα, IL6, and IL1), and gluconeogenesis (G6pc and GK) genes at GD17.5 in fetal liver exposed to 50 ppm benzene. However, this effect was not observed in fetuses exposed to 5 ppm benzene under the same regiment (Supplementary Figure 5). Altogether, these results reveal that benzene exposure during gestation can greatly impair glucose homeostasis, inflammatory responses and energy balance in adult offspring predisposing them to the development of metabolic syndrome.

DISCUSSION

This study is the first to demonstrate the severe whole-body metabolic imbalance in young adult offspring born to dams exposed to benzene during pregnancy at concentrations relevant to smoking. Metabolic imbalance in offspring was independent of body weight changes and was associated with impaired energy homeostasis and glucose metabolism, affecting both sexes, although the effect was more severe in adult male offspring. Our data provide a link between early life exposure to an environmental pollutant and the risk for developing metabolic syndrome later in life (in offspring). Further, it allows elucidating some of the risk factors for metabolic syndrome that pose a burden at the population level.

Benzene has been listed as an endocrine-disrupting chemical, but its association with the development of childhood metabolic disorders has not been studied in detail, especially when exposure occurs early in life. We show that gestational benzene exposure has no effect on the offspring’s body weight or body mass at weaning and up to 40 weeks; however, it is an indicator of insulin resistance and metabolic imbalance in adulthood. Adult male offspring exposed to benzene during gestation developed hyperglycemia and severe insulin resistance followed by increased β-cell mass and elevated expression of hepatic gluconeogenic genes. The effect of gestational benzene exposure on female offspring metabolic parameters became apparent only by 6-months of age, presented by hyperglycemia and mild insulin resistance with normal β-cell function. Both male and female offspring exhibited a considerable increase in hepatic gene expression associated with inflammation and ER stress. However, HOMA-IR the index of insulin resistance was elevated only in males but not in female offspring at 9 months of age. In support, we have recently demonstrated that adult female mice are completely resistant to the negative metabolic consequences of chronic benzene exposure that affects only male mice (Debarba et al., 2020). Benzene crosses the placenta and can be found in the placenta and fetuses immediately following exposure, exerting its effects on developing tissues and cells (Dowty et al., 1976). As such, our data identify gestation as a critical period for adult metabolic perturbations, with increased sensitivity of male offspring to the toxic effects of gestational benzene exposure. In contrast, the protective sex-specific effects in adult females observed in our recent study may have a hormonal basis (Piazza and Urbanetz, 2019); however, a causal relationship for these observations remains to be elucidated.

Glucose intolerance and insulin resistance displayed in offspring from benzene-exposed dams indicate that maternal benzene exposure has a strong influence on the offspring’s glucose metabolism likely by predisposing fetal organs involved in glucose disposal and insulin sensitivity to metabolic imbalance. Indeed, there was a significant proinflammatory response and an increase in genes associated with gluconeogenesis in the fetal liver exposed during pregnancy to 50 ppm benzene. This effect was not observed in the fetuses of pregnant females exposed to 5 ppm benzene under the same conditions. Indeed, the levels of exposure during pregnancy can differ, depending on the number of cigarettes taken by each pregnant woman, thus suggesting that fewer cigarettes lessen the susceptibility for metabolic risk in the offspring. Our data indicate a “window of sensitivity” in which maternal exposure to benzene causes inflammation and metabolic imbalance in the fetus that might be responsible for adverse outcomes including diabetes in adulthood.

Insulin secretion following glucose load was significantly higher in insulin-resistant male offspring from benzene-exposed dams, suggesting an adaptive β-cell response to compensate for insulin resistance, which is in line with increased β-cell mass in these animals. Interestingly, cigarette smoking during gestation induced changes in β-cell function including increased β-cell apoptosis and decreased β-cell mass in rodents (Bruin et al., 2007; Holloway et al., 2005). Islet changes and poor pancreatic β-cell viability were previously reported due to benzene exposure in rodents (Bahadar et al., 2015). The pancreas is considered to be especially susceptible to oxidative stress-mediated tissue damage (Lenzen et al., 1996). β-cell hypertrophy and hyperplasia occur during β-cell compensation to increase β-cell mass in response to hyperglycemia in diabetogenic states (Cerf et al., 2012). Similar to insulin-resistant and diabetic mice models, increased β-cell mass in male offspring can be likely attributed to β-cell hypertrophy and hyperplasia (Jones et al., 2010).

Both male and female offspring from benzene-exposed dams exhibited reduced parameters of energy homeostasis, including O₂, CO_2, and heat production. These findings suggest that gestational benzene exposure could alter the hypothalamic regulation of energy homeostasis in both sexes (Bouret and Simerly, 2004). Although 9-month-old male and female offspring display normal body weight, despite reduced energy and heat production, it is reasonable to hypothesize that during the aging process, such metabolic imbalance will ultimately affect the feeding and weight of these animals. In rodents, the hypothalamic energy balance-regulating system is structurally and functionally immature at the start of postnatal life, and the plasticity of the hypothalamic circuitry provides a route by which environmental signals can regulate energy homeostasis (Bouret, 2009, 2010). Our recent findings indicate that benzene exposure induces severe hypothalamic inflammation and ER stress in a sex-specific manner (Debarba et al., 2020). Perinatal exposure to persistent organic pollutants reduces energy expenditure with an associated increase in body weight in adult female offspring (La Merrill et al., 2014). In this regard, various studies have demonstrated the sex-specific effect of perinatal exposure to different pollutants and changes in energy homeostasis in the offspring (Heindel et al., 2017). Further research is needed to address the role of hypothalamic “programming” in response to gestational benzene exposure in both sexes.

It is important to note that our study used C57BL/6 mice for the animal model. The inbred mice display less trait variability thus allowing us the establishment of the exposure system. The use of inbred strains, by controlling interindividual variability, can reduce the number of animals needed in toxicological studies (Festing 2010). The outbred mice traditionally are considered to display high variability, thereby limiting their use in some studies; however, these animals can better represent the genetically diverse human population. Our study is the first to directly address the link between maternal exposure to benzene during pregnancy and severe metabolic imbalance in the offspring. Evaluating this effect in the outbred mice strains like genetically heterogeneous UM-HET3 mice should be addressed in future studies (Jackson et al., 1999).

Taken together, these data indicate that benzene exposure during gestation is the critical window to induce metabolic syndrome in the adult offspring, where male offspring are more sensitive to metabolic imbalance. Gestational exposure to benzene can interfere with epigenetic programming of gene regulation, thereby influencing the risk of diabetes development later in life. Thorough studies of genome-wide analysis will be needed to understand the epigenetic changes triggered by gestational benzene exposure. Such studies can improve our understanding of the possible maternal factors, underlying the programming of metabolic adversity across the life course of the offspring and identify the critical developmental period during which maternal exposure programs altered metabolic control in the offspring.

SUPPLEMENTARY DATA

Supplementary data are available at Toxicological Sciences online.

ACKNOWLEDGMENTS

The authors thank Madelynn Koch and Li Mao for technical assistance.

FUNDING

American Diabetes Association (1-lB-IDF-063), Center for Urban Responses to Environmental Stressors Center Grant (P30 ES020957), and Wayne State University startup funds for M.S.

DECLARATION OF CONFLICTING INTERESTS

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author notes

Lisa Koshko and Lucas K. Debarba contributed equally to this study.