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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: Toxicol Appl Pharmacol. 2022 Jan 3;436:115855. doi: 10.1016/j.taap.2021.115855

Whole Life Exposure to Low Dose Cadmium Alters Diet-Induced NAFLD

Jamie L Young a,b,c, Matthew C Cave a,b,d,e,f,g, Qian Xu h, Maiying Kong h, Jianxiang Xu c, Qian Lin c, Yi Tan a,c, Lu Cai a,c,d,*
PMCID: PMC8796138  NIHMSID: NIHMS1770190  PMID: 34990729

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a major global public health concern affecting more than 25% of the world’s population. Although obesity and diabetes are major risk factors for NAFLD, they cannot account for all cases, indicating the importance of other factors such as environmental exposures. Cadmium (Cd) exposure is implicated in the development of NAFLD; however, the influence of early-life, in utero Cd exposure on the development of diet-induced NAFLD is poorly understood. Therefore, we developed an in vivo, multiple-hit model to study the effect of whole-life, low dose Cd exposure on high fat diet (HFD)-induced NAFLD. Adult male and female C57BL/6J mice fed normal diets (ND) were exposed to 0, 0.5 or 5 ppm Cd-containing drinking water for 14 weeks before breeding. At weaning, offspring were fed ND or HFD and continued on the same drinking water regimen as their parents for 24 weeks. Cd exposure at different concentrations differentially altered HFD-associated adverse health effects, including liver injury. HFD-induced increased body weight, decreased glucose tolerance. Liver injury and lipid deposition were exacerbated by 5 ppm Cd exposure but attenuated by 0.5 ppm Cd exposure. Further, HFD blunted the response of metallothionein, a major Cd detoxification protein, in mice exposed to 5 ppm Cd but enhanced the response in mice exposed to 0.5 ppm Cd, suggesting a possible mechanism for Cd alteration of HFD-induced NAFLD. These results confirm the multi-hit nature of NAFLD and show whole life, low dose Cd exposure alters HFD-induced NAFLD with outcomes dependent on Cd concentration.

Keywords: Cadmium, Obesity, NAFLD, Whole-life Exposure, Metallothionein

Graphical Abstract

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1. Introduction

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease occurring in more than 25% of the world’s population (Rinella, 2015; Younossi et al., 2018). In the United States alone, NAFLD-related complications account for over 35,000 deaths annually (Paik et al., 2019). Unfortunately, the prevalence of NAFLD has almost doubled over the past 20 years and remains on the rise (Welsh et al., 2013) increasing in parallel with obesity (Williams et al., 2011; Chalasani et al., 2012). Although obesity is the primary risk factor for the development of NAFLD, approximately 20% of normal weight people develop the disease, indicating other risk factors are involved (Younossi et al., 2018). Moreover, multi-hit models have been proposed to explain the variability in NAFLD severity and affected patients (Takaki et al 2013; Buzzetti et al 2016; Peng et al 2020).

One such risk factor, or hit, is exposure to the non-essential metal cadmium (Cd). Importantly, the liver is a major target organ of Cd toxicity and accumulation (Arroyo et al., 2012). Cd is a naturally occurring heavy metal with no known biological function in humans. (Tinkov et al., 2017). Ranking number 7 on the Agency for Toxic Substances and Disease Registry list of environmental chemical hazards (ATSDR, 2012), Cd is one of the most common and detrimental metals present in our environment (Jacobo-Estrada et al., 2017), with a biological half-life between 4 and 37 years, depending on the organ (ATSDR, 2012). In the last century, exposure to Cd has dramatically increased (IPCS, 1992) due to its use in the production of batteries, pigments and plastics. The primary source of Cd exposure in the non-smoking, general population is ingestion of contaminated food and water, with an average daily intake of between 4 and 26 μg/day (Choudhury et al., 2001; Martorell et al., 2011; Kim et al., 2018).

Consistent with Cd’s bioaccumulation patterns, the liver is a target organ of Cd toxicity. In animal models of acute liver injury, Cd exposure induces inflammation, apoptosis and liver cell regeneration (Habeebu et al., 2000). These models, however, were designed to investigate the impacts of high Cd exposures and did not take into consideration the impact of lower, environmental exposures on liver disease. As such, more recent in vivo studies have revealed low dose exposure to Cd dysregulates NAFLD-related metabolic pathways, disrupts essential metal homeostasis and increases liver and serum triglycerides (Go et al., 2015; Zhang et al., 2015; Young et al., 2019). In addition, environmental Cd exposure has been associated with hepatic necroinflammation, NAFLD and NASH in men and necroinflammation women (Hyder et al., 2013). Despite these research advances, our understanding of Cd’s impact on the development and progression of NAFLD is unclear.

Our understanding of the potential interaction of Cd exposure with obesity is incomplete. Epidemiologically, the existing data associating Cd exposure with obesity are contradictory (Tinkov et al., 2017), varying between positive (Akinloye et al., 2010; Padilla et al., 2010) and negative associations (Tellez-Plaza et al., 2013; Nie et al., 2016) with other studies showing no significant association (Kelishadi et al., 2013; Gonzalez-Reimers et al., 2014). However, results from laboratory studies are more consistent, showing Cd exposure alone does not result in obesity (Levy et al., 2000; Fickova et al., 2003; Kawakami et al., 2013). Although it is well known that consumption of a high fat diet (HFD) contributes greatly to obesity, how exposure to low dose Cd influences initiation and progression of HFD-induced diseases is unclear (Zhang et al., 2020).

Another factor contributing to disease development and progression is that of chronicity and the timing. Originating in the 1990s, the Developmental Origins of Health and Disease (DOHaD) hypothesis suggests that exposures to environmental stressors during sensitive stages of human development (in utero and early childhood) increases susceptibility to adverse health outcomes in adulthood (Barouku et al., 2012; Gluckman et al., 2010; Thayer et al., 2012). Concern has risen about prenatal exposure to metals and the long-term adverse health implications due to the widespread production and use of metals, in parallel with the knowledge that metals can be passed from mothers to offspring via placenta and/or breast milk (Chen et al., 2014; Young et al., 2018). Researchers, including us, have just begun to develop models to study the impact of early life Cd exposure on disease development later in life, including hypertension (Hudson et al., 2019) and diet-induced cardiac hypertrophy (Liang et al., 2019). While results from these studies provide critical insight into the importance of early life exposure to Cd on the development of disease later in life, the impact on liver disease remains to be explored. Therefore, to address these key data gaps we developed an in vivo model to explore the hypothesis that whole life, low dose Cd exposure exacerbates HFD-induced NAFLD.

2. Materials and Methods

Animal models and exposures

Eight-week-old male (n=19) and female (n=51) C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were maintained on a 12-hour light/dark cycle at 25°C in a pathogen-free barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All experimental procedures were approved by the University of Louisville’s Institutional Animal Care and Use Committee, which complies with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). After one week of acclimation in the barrier facility, diets were switched from standard laboratory chow to purified ND (10% kcal fat: Research Diets D14020202, New Brunswick, NJ) to minimize the influence of metal contamination found in standard laboratory chow (Kozul et al., 2008). At 9 weeks old, mice were placed on a drinking water regimen of either tap water alone (control) or Cd containing water (0.5 or 5 ppm – final concentration). Cd containing drinking water was prepared weekly from stock solutions of CdCl2 (Alfa Aesar, Haverhill, MA) made with deionized water and stored at −80°C. Five ppm Cd was used as a positive control. Based on a survey of the literature, 5 ppm Cd is one of the lower concentrations of Cd tested with results supportive of metabolic syndrome phenotypes. In addition, we used a ten times lower concentration of 0.5 ppm Cd. According to the ATSDR ~100 to 300 mg/kg Cd (1 ppm = 1 mg/kg) is lethal to 50% of rodents after acute oral exposure (ATSDR, 2012). Our oral exposures are less than 1% and 0.1% of this Cd level.

After 14 weeks, mice were placed into mating groups (F0) (1 male to 2 or 3 females) within each drinking water exposure (Figure 1A, B). Pregnant dams continued on the same drinking water regimen through weaning. At weaning, male offspring (F1) were either fed the same ND as their parents or a HFD (60% kcal fat: Research Diets D14020205, New Brunswick, NJ) and continued on the same drinking water regime as their parents. Epidemiologically, males develop NAFLD more often and with greater severity than pre-menopausal women, and this outcome is recapitulated in rodent studies (Hyder et al., 2013; Lonardo et al., 2019). Therefore, we focused on male offspring in our study. Food and deionized water were provided ad libitum. Body weights and water consumption were recorded weekly.

Figure 1.

Figure 1.

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Model of whole life exposure to Cd and HFD. (A) Adult male and female C57BL/6J mice on defined diets were exposed to control drinking water, or water containing 0.5 or 5 ppm Cd for 14 weeks before being established into breeding pairs. Pregnant dams and male offspring were continuously exposed and continued on the same drinking water regime as their parents after weaning. At weaning, male offspring were also fed either a normal or high-fat diet (ND or HFD, respectively) for 24 weeks. (B) One week prior to sacrifice, IPGTT tests were performed and body composition determine by DEXA scan (DS). Body weight was measured once a week for 24 weeks starting at weaning, through sacrifice. Weekly body weight in (C) ND male mice and (D) HFD mice. IPGTT = intraparietal glucose tolerance test, wks = weeks. Results are reported as mean ± SEM (n=4-6) for body weights.

Mice were anesthetized with an intraperitoneal injection of avertin (250 mg/kg). Blood was collected from the inferior vena cava prior to euthanasia via exsanguination. For each mouse, liver weight was recorded, and portions of liver tissue were snap-frozen in liquid nitrogen, processed for RNA isolation, fixed in 10% neutral buffered formalin for histology and immunohistochemistry, frozen-fixed in Tissue Tek OCT-Compound (Sakura Finetek, Torrance, CA) or used for metals analysis. The liver weight to tibia length ratio (liver weight in grams divided by tibia length in millimeters) was used as an index of liver size changes.

Glucose tolerance test

The intraperitoneal glucose tolerance test (IPGTT) was used to measures the ability to clear an injected load of glucose. Mice were fasted for 6 hours, weighed and injected intraperitoneally with a 2 g/kg glucose solution. Blood glucose levels were measured at 0 (pre-injection), 15, 30, 60, and 120 minutes post-glucose injection using a FreeStyle complete blood glucose monitor system (Abbott Diabetes Care Inc., Alameda, CA). A time course of absolute blood glucose measurement and area under the curve (AUC) were determined for each animal.

Biochemical analysis

Plasma insulin levels were measured with the Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem, Elk Grove Village, IL) using the manufacturer’s instructions for preparing samples and reagents for the wide range assay (0.1 - 12.8 ng/mL). Levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), clinical biomarkers of hepatic injury, were determined spectrophotometrically using Infinity™ ALT (GPT) and AST (GPO) Liquid Stable Reagents (Thermo Fisher Scientific, Waltham, MA) per the manufacturer’s instructions. Hepatic triglycerides (TG) and total hepatic cholesterol (TC) were determined spectrophotometrically using Infinity™ Triglycerides Liquid Stable Reagents (Thermo Fisher Scientific, Waltham, MA) per the manufacturer’s instructions.

Histological analysis

Paraffin-embedded liver sections (5 μm) were deparaffinized with xylene, rehydrated with graded ethanol washes and stained with hematoxylin and eosin (H&E) to assess overall hepatic structure. Pictures were taken on an Olympus BX43 microscopy system using Olympus cellSense Imaging Software (Shinjuku City, Tokyo, Japan). Steatosis was scored as percent of hepatocytes in a 100x field containing fat (<25% = 1+; <50% = 2+; <75 = 3+; >75% = 4+) (Nanji et al., 1989). For each animal, ten 100x fields were scored. Histological evaluation of neutral lipids was done using oil red O (ORO) (C26H24N4O) (Sigma Aldrich, St. Louis, MO) (Mehlem et al., 2013). Fresh frozen liver sections (10 μm) were fixed in 10% neutral buffered formalin for 20 minutes and stained with ORO for 30 minutes at room temperature (Mehlem et al., 2013). The same day, ten 100x pictures were taken on an Olympus BX43 microscopy system using Olympus cellSense Imaging Software (Shinjuku City, Tokyo, Japan). Exposure time and gain settings were determined using a sample known to have excessive ORO staining. Image J software (National Institutes of Health, Bethesda, Maryland) was used to quantify the percent of area positively stained with ORO.

Total RNA isolation, cDNA synthesis and qRT-PCR

Total RNA was isolated from liver tissues using STAT 60 (Amsbio LLC, Abingdon, United Kingdom), following the manufacturers protocol for RNA isolation. Quantitative RT-PCR reactions were performed using TaqMan® RNA assays (Thermo Fisher Scientific, Waltham, MA). TaqMan gene expression assays (GAPDH, Mm99999915_g1; MT1, Mm00496660_g1; MT2, Mm04207591_g1) were combined with Entrans 2x qPCR Probe Master Mix (ABclonal, Woburn, MA) and DEPC water. For all qRT-PCR runs a “no cDNA control” was included. Reactions were performed on a LightCycler® 96 Real-Time PCR Cycler (Roche, Basel, Switzerland).

Protein extraction and Metallothionein (MT) Western blot

Liver tissues (~30 mg/liver) were homogenized on ice in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM 0.5 EDTA (pH 8.0), 50ug/mL phenylmethylsulfonyl fluoride and Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO). Homogenized samples were placed on a rotating mixer for 4 hours at 4°C and centrifugation at 12,000 rpm at 4°C for 20 minutes. Protein concentrations were quantified by the Bradford assay (Bio-Rad protein assay dye reagent; Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as the reference protein.

MT expression was detected with a modified Western blot protocol as previously described, with slight modifications (Wang et al., 2006; Liang et al., 2019). The transfer conditions for MT are not appropriate for the detection of β-actin; therefore, 2 parallel gels are run under the same conditions except the gel for MT was transferred to a nitrocellulose membrane in a 15% methanol, calcium chloride containing buffer while the β-actin gel, run in parallel, was transferred in a 20% methanol buffer without calcium chloride, as described before (Wang et al., 2006; Liang et al., 2019).

Metals analysis

Each liver sample (20-100 mg wet-weight) was digested in 1 mL of 70% concentrated trace metal grade nitric acid in an 85°C water bath for 4 hours. After digestion, samples were cooled to room temperature, filtered with a 100 μm filter and brought to a final concentration of nitric acid to 2% with Milli-Q deionized water. Metal levels were assessed using an X Series II quadrupole inductively coupled plasma mass spectrometry (ICP-MS) (Thermo Fisher Scientific) equipped with an ESI SC-2 autosampler (Elemental Scientific, Inc.) for sample injection. During sample injection, internal standards including Bi, In, Li, Sc, Tb and Y (Inorganic Ventures) were mixed with each sample for drift correction and accuracy improvement. Each sample was analyzed three times and metal levels calculated and presented as ng/g wet tissue. Anything less than the Intercept Concentration was considered non-detectable.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 7 statistical software (GraphPad Software Inc., San Diego, CA) and R statistical software version 3.6.2. Results are first reported as the mean ± standard deviation (SD; n=3-6), and then followed by two-way ANOVA analysis along with post-hoc t-tests. Two-way ANOVA was used to determine if there was significant diet effect, Cd effect, and the interaction between diet and Cd exposure. If one of the main effects or interaction were significant, then Bonferroni’s adjusted post hoc t-tests were performed to compare Cd effect within a diet group (* indicating significant Cd effect compared to group control, # indicating significant Cd effect compared to Cd 0.5 ppm dose within diet group), and to compare the diet effect within a Cd group (@ indicating significant diet effect compared to corresponding ND). For body weight, repeated measures analysis of variance (rANOVA), a special case of linear mixed effect models, was used. In the rANOVA, the main effects (Cd exposure, diet, time in weeks) and their interactions as fixed effects, and subject as random effects were included. The estimated fixed effects, their standard errors, and p-values were used for statistical inferences. Results are reported as mean ± SEM (n=4-6) for body weights. A test is claimed significant if its p-value is less than 0.05.

3. Results

Body weight

As expected, mice fed HFD gained significantly more weight over time (p <0.001) (Figure 1C,D). Weight gain in ND-fed mice exposed to 5 ppm Cd was slightly reduced compared to ND and 0.5 ppm exposed mice, although not statistically significant. (Figure 1C). HFD-fed mice exposed to 5 ppm Cd gained significantly more weight compared to their corresponding HFD-fed controls (p <0.001), while HFD-fed mice exposed to 0.5 ppm Cd had reduced weight gain compared to their corresponding controls (p <0.001) (Figure 1D). These data suggest 5 ppm Cd exacerbates, but 0.5 ppm Cd reduces HFD-induced weight gain.

Hepatic Cd levels and weekly administered Cd intake

Cd accumulated in the liver in a concentration-dependent manner independent of diet (Figure 2A). HFD consumption did not influence Cd accumulation in control or 5 ppm exposed mice; however, HFD-fed mice exposed to 0.5 ppm Cd tended to have greater hepatic Cd content compared to the corresponding ND-fed mice.

Figure 2.

Figure 2.

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Hepatic Cd levels and weekly Cd intake. This figure shows Cd accumulated in the liver in a concentration-dependent manner, reflecting the average Cd intake delivered by drinking water. (A). Hepatic Cd levels measured by ICP-MS. (B) Average weekly Cd intake delivered by drinking water in ND mice. (C) Average weekly administered Cd intake delivered by drinking water in HFD mice. *, p < 0.05 compared to group control; #, p < 0.05 compared to Cd dose within diet group.

Mice were exposed to Cd in their drinking water, therefore we measured water consumption and calculated the average administered Cd intake (Figure 2B and C). Over the course of the study, 0.5 and 5 ppm Cd exposed mice drank an average of 5.15 ± 1.45 and 6.13 ± 1.45 mL of water per a day, respectively, compared to controls who drank an average of 4.60 ± 0.55 mL of water per a day. Thus, treated mice drank similar amounts of water compared to controls, independent of diet. The average daily dose of Cd in mice that drank water with 0.5 ppm Cd was 2.58 ± 0.62 and 2.87 ± 0.71 μg in ND and HFD fed mice, respectively. In mice that drank water with 5 ppm Cd the average daily dose was significantly greater: 30.67 ± 7.27 (p <0.0001) and 24.53 ± 5.42 μg (p < 0.0001) in mice fed ND and HFD mice, respectively. Interestingly, in 5 ppm Cd exposed mice fed HFD, Cd intake was significantly decreased (p = 0.012) compared to exposed, ND-fed mice.

IPGTT and plasma insulin

Diets high in fat are a major risk for insulin resistance (Park et al., 2001; Winzell et al., 2004). Increased blood sugar levels in combination with increased insulin in the blood are indicators of insulin resistance (Bowe et al., 2014), a hallmark of NAFLD that greatly influences disease progression (Manco, 2017). Mice fed HFD had significantly increased blood glucose levels compared to ND-fed animals (Figure 3A and B) with a trend towards increased insulin in the plasma (Figure 3D) suggesting the development of diet-induced insulin resistance. In ND-fed mice, Cd exposure did not significantly alter glucose tolerance (Figure 3A and C). Similarly, 5 ppm Cd exposure did not significantly impact HFD-induced impairment of glucose clearance (Figure 3B and C). However, mice exposed to 0.5 ppm Cd paradoxically showed improved glucose clearance and reduced plasma insulin levels similar to those observed in ND-fed animals (Figure 3D).

Figure 3.

Figure 3.

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Effects of whole life exposure to Cd and HFD on glucose handling and plasma insulin. Whole life exposure to Cd alters glucose clearance and plasma insulin in HFD mice. Blood glucose levels in (A) ND and (B) HFD mice after IPGTT, performed one week prior to sacrifice. (C) Integrated area under the curve (AUC) showing quantitative changes in blood glucose levels after glucose challenge. (D) Insulin levels in plasma at time of sacrifice. Results are reported as the mean ± SD (n=4-6). *, p < 0.05 compared to group control; #, p < 0.05 compared to Cd dose within diet group; @, p < 0.05 compared to corresponding ND.

Hepatic injury and NAFLD

Mice fed HFD tended to have enlarged livers (Figure 4A) and increased ALT, but not AST, levels compared to ND-fed mice (Figure 4B and C), although not statistically significant. HFD-fed mice exposed to 5 ppm Cd had significantly larger livers compared to those fed ND and compared to HFD-fed controls, indicating that exposure to 5 ppm Cd significantly exacerbated HFD-induced hepatomegaly (Figure 4A). In contrast, 0.5 ppm Cd reduced the size of the HFD-enlarged livers, down to levels seen in ND-fed mice (Figures 4A). Interestingly, independent of diet, exposure to 5 ppm Cd caused liver injury as indicated by increased ALT and AST levels, suggesting exposure to 5 ppm Cd alone inflicts damage to the liver (Figure 4B and C).

Figure 4.

Figure 4.

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Effects of whole life Cd exposure and HFD on liver injury. Cd exposure changes liver size in HFD mice and exposure to 5 ppm Cd increases liver transaminases, independent of diet. (A) Ratio of liver weight in grams to tibia length in millimeters, a measure of hepatomegaly. (B) Plasma aspartate aminotransferase (AST) and (C) plasma alanine aminotransferase (ALT) activity. Results are reported as the mean ± SD (n=4-6). *, p < 0.05 compared to group control; #, p < 0.05 compared to Cd dose within diet group; @, p < 0.05 compared to corresponding ND.

Histological analysis of liver tissue revealed significant increases in steatosis in mice fed HFD compared to controls as seen in representative photomicrographs of H&E (Figure 5A) and oil red O-stained liver tissue (Figure 5B) as well as in the quantification of the histology (Figure 5C and D). Additionally, biochemical analysis of liver tissue showed HFD fed mice had greater hepatic triglyceride levels compared to mice fed ND (Figure 5E).

Figure 5.

Figure 5.

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Effects of whole life Cd exposure and HFD on NAFLD. Exposure to 5 ppm Cd exacerbates HFD-induced steatosis while exposure to 0.5 ppm Cd rescues HFD-induced steatosis. (A) Representative photomicrographs of paraffin embedded liver tissue stained with hematoxylin & eosin staining (H&E, 200x). (C) Steatosis was scored as percent of liver cells in 5, 10x fields per liver containing fat. (B) Representative photomicrographs of Oil Red O (neutral lipids, ×200) stained frozen liver sections. (D) Image analysis of ORO-positive staining was performed using Image J, and results are shown as percentage of microscope field. (E) Hepatic triglyceride (TG) levels. (F) Total hepatic cholesterol levels. Results are reported as the mean ± SD (n=4-6). *, p < 0.05 compared to group control; #, p < 0.05 compared to Cd dose within diet group; @, p < 0.05 compared to corresponding ND.

Cd exposure did not cause either histological steatosis or liver injury in ND-fed mice according to liver morphology (Figure 5A and C) and lipid deposition (Figure 5B and D). However, Cd exposure significantly impacted these measures in HFD-fed mice. Specifically, 5 ppm Cd exacerbated HFD-induced steatosis and biochemical analysis of hepatic TG content confirmed these results (Figure 5E). However, hepatic TC levels remained unchanged (Figure 5F).

Similar to 5 ppm Cd, exposure to 0.5 ppm Cd did not overtly impact liver morphology or lipid deposition in ND-fed mice. However, the outcomes in HFD-fed mice after 0.5 ppm Cd continued to be very different from 5 ppm Cd. In fact, exposure to 0.5 ppm Cd reduced the pathology to levels seen in ND-fed mice (Figure 5A–D). Taken together these data indicate exposure to 5 ppm Cd exacerbates HFD-induced NAFLD while exposure to 0.5 ppm Cd attenuates the diet-induced hepatic pathology.

Metallothionein (MT)

MT, a small, low molecular weight, cysteine rich protein, plays a major role in protecting the body from Cd toxicity and the expression of MTs generally increases with elevations in tissue Cd levels (Klaassen et al., 2009). There are 4 major mammalian isoforms of MT, of which MT-1 and MT-2 are highly expressed in the liver (Klaassen et al., 1999). Therefore, we focused on determining the response of MT in protection of the liver against Cd accumulation and toxicity by measuring hepatic MT protein and mRNA (both MT-1 and MT-2) levels.

In mice fed ND, exposure to 5 ppm Cd resulted in a significant increase in mRNA levels of MT compared to controls, a result not observed in 0.5 ppm exposed mice (Figure 6A and B). HFD alone did not alter hepatic mRNA levels of MT; however, there was a slight increase in MT mRNA levels in HFD-fed mice exposed to 0.5 ppm Cd. In contrast, in HFD-fed mice exposed to 5 ppm Cd, MT mRNA expression significantly decreased compared to those fed ND.

Figure 6.

Figure 6.

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Effects of whole life Cd exposure and HFD on metallothionein (MT). Cd exposure alters MT. (A) Hepatic mRNA expression of MT-1 and (B) MT-2. (C) Representative western blot for hepatic MT in control mice, (D) Cd exposed mice (NOTE: membrane cropped to remove loading control lane at front of the blot), and (E) corresponding densitometric analyses shown as fold of control. GAPDH was used as a loading control. Results are reported as the mean ± SD (n=3-6). *, p < 0.05 compared to group control; #, p < 0.05 compared to Cd dose within diet group; @, p < 0.05 compared to corresponding ND.

In the liver, MT protein levels showed a similar pattern to that of MT mRNA. Panel C (Figure 6) shows representative western blots containing samples for each of the six experimental groups and Panel D (Figure 6) shows quantification of the western blot. As was seen with mRNA, HFD did not change hepatic MT protein levels. In ND-fed animals, exposure to 5 ppm Cd significantly increased MT protein levels compared to control and 0.5 ppm Cd exposed mice, which was consistent with the mRNA level results. By contrast, for HFD animals, 0.5 ppm Cd significantly increased MT protein levels while 5 ppm Cd significantly decrease levels. These outcomes were consistent with the mRNA data.

4. Discussion

For the present study we developed an in vivo model to study the hypothesis that whole life exposure to low dose Cd exacerbates HFD-induced NAFLD. We found exposure to Cd alone, starting in utero and continuing for 24 weeks post weaning, did not induce NAFLD. However, in HFD-fed offspring we found major contrasting outcomes based on Cd exposure levels.

Although Cd burden is associated with liver-related mortalities and NAFLD (Hyder et al., 2013; Li et al., 2021) Cd exposure in our study did not induce NAFLD in ND-fed mice, though we did find increased levels of liver transaminases (ALT and AST) in Cd exposed mice independent of diet. Our data conflict with the only other previous in vivo report of Cd and NAFLD, which reported Cd exposure induced hepatotoxicity and dysregulated NAFLD-related metabolic pathways in ND-fed mice (Go et al., 2015). That study exposed 8-week-old male mice for 20 weeks to 10 ppm Cd in drinking water. Consistent with our study, they found both ALT and AST were elevated, but they also found liver triglycerides and lipid deposition were also elevated, which were not found in our study. The difference is most likely due to the fact that the Cd exposure they used was twice the exposure we used in our study. This possibility would suggest the changes in ALT and AST may precede the changes in lipid deposition and hepatic triglycerides following Cd exposure. Heavy metals, and Cd in particular, are associated with elevated ALT and AST activity (Kang et al., 2013; Rao et al., 2017) and these elevations have been found in otherwise healthy adults (Kang et al., 2013). Therefore, it is possible that the levels of Cd in both studies were sufficient to increase transaminase levels, but in our study, the lower concentration of Cd was not sufficient to cause progression to NAFLD.

It is also notable that mice in our study were exposed to Cd starting in utero whereas in the study by Go et al (2015) mice were not exposed to Cd until they were 8 weeks old as the authors stated in their methods that they specifically chose 8-week-old mice as their intention was not to assess developmental toxicity. Thus, it is possible the chronicity of the exposure may have played a role in these different outcomes. For example, it is well documented that prenatal Cd exposure is associated with altered DNA methylation patterns (Castillo et al., 2012; Kippler et al., 2013; Mohanty et al., 2015). Castillo et al. (2012) reported altered methylation patterns of the hepatic glucocorticoid receptor, a phenomenon linked to increased risk of cardiometabolic disorders in adulthood. However, the consequences of these early life, Cd-induced altered DNA methylation patterns on the development of diseases later in life is largely unknown. Therefore, an interesting future course for our model would be to investigate the impact of early-life Cd exposure and altered DNA methylation patterns on the development of HFD-induced NAFLD.

Supporting our hypothesis that Cd exposure exacerbates HFD-induced NAFLD, exposure to 5 ppm Cd enhanced HFD-induced weight gain, insulin resistance, hepatic injury (hepatomegaly and plasma transaminase) and steatosis. Although similar results have been reported with exposures to polychlorinated biphenyls and vinyl chloride (Wahlang et al 2013; Lang et al 2018), where exposure to the environmental contaminate alone did not overtly cause liver injury but combined with HFD resulted in exacerbated hepatic steatosis, we are the first to report these outcomes with Cd exposure. In contrast, and to our surprise, exposure to 0.5 ppm Cd reduced NAFLD induced by HFD as evidenced by a marked decrease in body weight, improved glucose handling and decreased insulin in the plasma, similar to levels seen in ND-fed animals as well as diminished HFD-induced steatosis.

The literature in support of Cd as a therapeutic is virtually non-existent. However, there are a few studies that have exploited Cd’s adverse effects on cell proliferation and the immune system in the treatment of rheumatoid arthritis (Ansari et al., 2015; Bonaventura et al., 2017). Interestingly, Ansari et al (2015) showed in female rats between 6-8 weeks old, exposure to 5 ppm Cd chloride in drinking water for 21 days restored antioxidant levels, arrested progression of the autoimmune disease and down-regulated pro-inflammatory modulators whereas exposure to 50 ppm Cd chloride had the complete opposite effect, increasing the pro-inflammatory response and exacerbating the disease. These results are particularly interesting in that the outcomes at one concentration of Cd are very different than the outcomes at a 10 times greater concentration – a phenomenon similar to what we observed in our study. Therefore, in our model, the effects of inflammation on NAFLD progression are an important future consideration. To be clear, we are not proposing 0.5 ppm Cd as a therapy for NAFLD.

The capacity of Cd to mimic essential metals may, in part, explain the preventative effect of 0.5 ppm Cd in HFD-induced NAFLD. Obese individuals have high rates of micronutrient deficiency, despite the excessive intake of food, and such deficiencies may contribute to the development of HFD-induced disease (Garcia et al., 2009; Via et al., 2012). It is possible that exposure to Cd at this lower concentration of 0.5 ppm sufficiently acts to seemingly restore the micronutrient deficiency through molecular mimicry and thus reduce the effect of HFD. However, further studies would need to be conducted to examine micronutrient status in these animals as well as the long term, later life implications for such an exposure.

In our model we found a strong effect on MT that may begin to explain the contrasting, Cd level dependent hepatic outcomes. MT, a small, low molecular weight, cysteine rich protein, plays a major role in protecting the body from Cd toxicity and the expression of MTs generally increases with elevations in tissue Cd levels (Klaassen et al., 2009). As expected, exposure to 5 ppm Cd increased both MT mRNA and protein levels in mice fed normal diet. However, in HFD-fed mice, 5 ppm Cd exposure dramatically lowered MT response compared to ND-fed mice, indicating a possible interaction between HFD and Cd that hinders the protective response of MT, suggesting a possible mechanism by which exposure to Cd may exacerbate HFD-induced liver disease. Literature reporting MT knockout mouse strains are more susceptible to HFD-induced effect supports this idea (Lindeque et al., 2015; Gu et al., 2107). Thus, studies using liver specific MT-overexpressing and KO mice would be important future directions.

In contrast, exposure to 0.5 ppm Cd did not change MT mRNA or protein levels normal diet fed mice but did increase these measures in HFD fed mice. As MT plays a major role in essential metal homeostasis, its induction may help elevate HFD-associated nutrient deficiencies and thus prevent obesity related diseases. Indeed, animal studies implicate MT in obesity prevention, but the mechanisms remain unclear (Sato et al., 2013; Byun et al., 2011; Lindeque et al., 2015; Kawakami et al., 2019).

While studies have looked at the impact of diet and Cd exposure as independent variables, our study is among the first to investigate how these two factors may act together to influence initiation and progression of NAFLD. Our data show that exposure to 5 ppm Cd exacerbated HFD-induced NAFLD and exposure to 0.5 ppm Cd reversed the phenotype. While we have shown that altered MT levels, both mRNA and protein, may contribute to the contrasting outcomes further studies are needed to elucidate the underlying mechanisms. In particular, the mechanism of Cd-associated disease recovery undoubtedly deserved further investigation. As such, future studies may include investigating the role of Cd and inflammation in NAFLD pathogenesis.

The current study is one of the first to investigate the impact of whole life Cd exposure (in utero throughout adulthood) on the development HFD-induced NAFLD, taking into consideration that environmental exposures can be life-long, spanning multiple windows of susceptibility. However, the potential impact of Cd exposure in utero is unable to be precisely dissected with this exposure paradigm and warrants follow-on studies.

While the results from this study provide critical insight into the “multiple-hit” nature of NAFLD, considering diet and environmental exposures as contributing variables this study only investigates the impact of two concentrations of Cd in disease outcome, thus not providing an informative dose response curve. Additional Cd exposures would possibly provide valuable insight into non-monotonic response to Cd observed in our model. Furthermore, while data suggests a role for MT in Cd-exacerbated, HFD-induced NALFD, the mechanism remains unknown and the interaction between HFD and Cd requires further investigation.

Highlights.

  • Whole life low-dose Cd exposure alone, starting in utero, did not cause NAFLD

  • As expected, 24-week HFD feeding post-weaning caused NAFLD in mice

  • In mice fed HFD, whole life exposure to 5 ppm Cd exacerbated HFD-induced NAFLD

  • In mice fed HFD, whole life exposure to 0.5 ppm Cd rescued HFD-induced NAFLD

  • Differences in MT response may explain the contrasting hepatic outcomes

Acknowledgements:

This work was supported by the National Institutes of Health [T32ES011564, R35ES028373, P30ES030283, R01ES032189, P42ES023716, R21ES031510, P20GM113226]; the American Diabetes Association [1–18-IBS-02]. Dr. Jamie Young would like to acknowledge the contributions of several members of his doctoral dissertation committee who were not included as coauthors who provided valuable feedback and guidance throughout this study (Drs. J. Christopher States, Michael L. Merchant, ShaoYu Chen). Drs. Bin Zhou, Wenqian Zhou, Li Zhuo and Hongbo Men are also acknowledged.

Footnotes

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Authorship (CRediT) Statement:

Jamie L. Young: Conceptualization, Visualization, Project administration, Investigation, Writing - Original Draft

Qian Xu: Formal Analysis, Writing – Review and Editing

Maiying Kong: Formal Analysis, Writing – Review and Editing

Jianxiang Xu: Resources, Investigation Writing – Review and Editing

Qian Lin: Resources, Writing – Review and Editing

Yi Tan: Writing – Review and Editing

Matthew C. Cave: Writing - Review & Editing, Supervision

Lu Cai: Methodology, Writing - Review & Editing, Supervision

Declarations of interest: none

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