Abstract

   Trichloroethylene (TCE) and inorganic arsenic (iAs) are environmental
   contaminants that can target the kidney. Chronic exposure to TCE is
   associated with increased incidence of renal cell carcinoma, while
   co-exposure to TCE and iAs likely occurs in exposed human populations,
   such as those near Superfund sites. In order to better understand the
   kidney health consequences of TCE and/or iAs exposure, a genetically
   heterogeneous mouse population derived from FVB/NJ and CAST/EiJ mouse
   strains and deficient for multidrug resistance genes (Abcb1a^tm1Bor,
   Abcb1b^tm1Bor) was chronically exposed for 52-weeks to varying
   concentrations of TCE and iAs. Although no exposure group resulted in
   primary renal cell tumors, kidneys from exposed mice did have
   significant increases in histologic and biochemical evidence of renal
   tubular disease with each toxicant alone and with combined exposure,
   with males having significantly higher levels of damage. Although no
   added increase in tubular disease was observed with combination
   exposure compared to single toxicants, molecular changes in kidneys
   from mice that had the combined exposure were similar to those previous
   observed in an embryonic stem cell assay for the P81S TCE-induced renal
   cell carcinoma mutation in the Von Hippel-Lindau syndrome (VHL) gene.
   While this model more accurately reflects human exposure conditions,
   development of primary renal tumors observed in humans following
   chronic TCE exposure was not reproduced even after inclusion of genetic
   heterogeneity and co-carcinogenic iAs.

   Keywords: renal toxicity, trichloroethylene, arsenic, environmental
   exposure
     __________________________________________________________________

   Trichloroethylene (TCE) is a chlorinated solvent that was widely used
   as an industrial degreaser and dry-cleaning agent. Due to its wide use
   and improper disposal, TCE has been found at the majority of the
   current and proposed National Priority List (Superfund) sites
   ([30]Scott and Cogliano 2000), and is the most commonly reported
   organic groundwater contaminant ([31]National Research Council (U.S.)
   Committee on Human Health Risks of Trichloroethylene. 2006). Exposure
   to TCE has been associated with a variety of cancers and disorders,
   including renal cancers and renal injury ([32]Liu et al. 2010;
   [33]Wartenberg et al. 2000) and has been associated with a specific
   mutation in the Von Hippel-Lindau syndrome (VHL) gene ([34]Brauch et
   al. 1999). Functional tests of the TCE-associated VHL P81S mutation in
   an embryonic stem cell-based assay showed that it elicits unique
   characteristics not observed with other VHL mutations including
   diversified cellular metabolism, resistance to apoptosis, and reduced
   Ataxia-Telangiesctasia mutated (ATM) response to DNA damage
   ([35]Desimone et al. 2013).

   There has recently been increased interest and study on the effects of
   toxicant mixtures on health outcomes ([36]Kapraun et al. 2017;
   [37]Pollock et al. 2017), particularly for toxicants like TCE whose
   effects on disease severity and outcome may be mediated by co-exposures
   in the environment. Inorganic arsenic (iAs) is a prime candidate for
   studying interactions of multiple toxicants since it is a commonly
   encountered environmental contaminant found in soil and groundwater
   secondary to anthropogenic activities including mining, farming, and
   fossil fuel combustion ([38]National Toxicology Program 2016;
   [39]Naujokas et al. 2013). Exposure to iAs is associated with adverse
   health effects in humans including development of chronic kidney
   disease from chronic exposure to arsenic ([40]Diyabalanage et al. 2017;
   [41]Hsu et al. 2017; [42]Zheng et al. 2015). In addition to drinking
   water, iAs exposure may occur through foods and processed beverages
   such as rice grown in areas with high iAs levels ([43]Naujokas et al.
   2013; [44]Davis et al. 2012; [45]Gilbert-Diamond et al. 2011;
   [46]Jackson et al. 2012; [47]Meharg et al. 2008). Chronic exposure to
   iAs at levels higher than the current EPA maximum contaminant level (10
   μg/L) are estimated to occur in greater than 3 million people in the
   United States ([48]Naujokas et al. 2013) and it is reasonable to expect
   that at least some of the people exposed to iAs will also be exposed to
   other environmental toxicants like TCE.

   Co-exposure to potentially carcinogenic toxicants may result in
   enhancement of carcinogenesis or confound retrospective studies on the
   effects of a single toxicant. There is increasing evidence that iAs may
   act as a co-carcinogen or promoter in combination with various other
   toxicants ([49]Germolec et al. 1997; [50]Germolec et al. 1998;
   [51]Rossman 2003; [52]Rossman et al. 2001; [53]Rossman et al. 2002;
   [54]Wang et al. 2002; [55]IARC Working Group on the Evaluation of
   Carcinogenic Risks to Humans 2012). Due to the presence of iAs in food
   and its natural or enhanced presence in the environment, certain areas
   of the United States have higher exposure levels. This includes areas
   of North Carolina in the “slate belt” which overlaps with a Superfund
   site at Camp Lejeune, an area also known to have high levels of TCE
   contamination ([56]Bove et al. 2014; [57]Sanders et al. 2012),
   illustrating the need for research into the potential effects of
   co-exposure to these toxicants. Onslow County, home to Camp Lejeune had
   renal cancer incidences of 21.3 per 100,000 from 2008-2012, well above
   the national average (Centers for Disease Control and Prevention).
   While studies of exposed human populations provide the strongest link
   between toxicant exposure and disease, use of appropriate rodent models
   can provide evidence of toxicity and strengthen the case for human
   health effects when many epidemiological confounds exists.

   The most recent National Toxicology Program (NTP) carcinogenesis study
   on TCE used a single dose in mice based on the low dose from a
   preceding 13-week study ([58]National Toxicology Program 1990).
   Survival of TCE-exposed male mice was lower than that of controls, and
   cytomegaly was reported in male and female TCE-exposed mice, but none
   of the vehicle-exposed mice. Increased incidence of hepatocellular
   tumors was observed in mice, but no renal tumors were observed. While
   the classic NTP approach has some advantages, it also has substantial
   limitations. Due to the lack of genetic diversity in the models, these
   models do not capture the variation in response to toxicants that is
   due to genetic heterogeneity in humans they seek to model.

   In this study, we utilized two common environmental toxicants, TCE and
   iAs, in doses designed to mirror human exposure levels to model
   combination environmental exposures, used a genetically heterogeneous
   population of mice to capture genetic variability, and fed a diet whose
   nutrient profile is similar to the typical western diet to better model
   the nutritional environment in which these toxicants would have their
   effects in the US.

Materials and Methods

Animals

   All housing conditions and procedures were approved by the
   Institutional Animal Care and Use Committee. An F3 mouse population was
   derived from two phylogenetically distant inbred mouse strains. The Mus
   musculus domesticus inbred strain FVB/N-Abcb1a^tm1Bor, Abcb1b^tm1Bor
   that has mutations in the multi-drug resistance (MDR) transporter genes
   Abcb1a and Abcb1b, resulting in loss of function of the MDR
   transporter. Mice have an especially active MDR system, while
   Abcb1a^tm1Bor, Abcb1b^tm1Bor double homozygous mutant mice exposed to
   arsenic have increased sensitivity to acute arsenic toxicity compared
   to wild-type mice, and higher arsenic accumulation in tissues including
   the kidney ([59]Liu et al. 2002), better modeling that observed in
   human tissues. The M. m. castaneus inbred strain CAST/EiJ is a
   wild-derived strain that is genetically distinct from the FVB/N strain
   with a highly divergent polymorphism profile.

   Breeding of mice was performed in house at North Carolina State
   University, Raleigh, NC. The breeding colony and study mice were
   maintained in a temperature-controlled environment at 21+/− 2° on a
   12-hour light: 12-hour dark schedule. Female FVB/N-Abcb1a^tm1Bor,
   Abcb1b^tm1Bor mice (Taconic Biosciences) were crossed with male
   CAST/EiJ mice (Jackson Laboratory) to create an F1 population that was
   intercrossed to create an F2 mouse population. Because only one-quarter
   of mice in this generation were expected to be Abcb1a^tm1Bor,
   Abcb1b^tm1Bor double homozygous mutant and a large number of mice with
   this genotype were required for the study population, F2 mice were
   genotyped to identify mice homozygous for the double knockout of the
   MDR transporter as previously reported ([60]Schinkel et al. 1997).
   These mice were then intercrossed to produce the study population of F3
   mice homozygous for Abcb1a^tm1Bor, Abcb1b^tm1Bor.

Toxicant Exposure

   One hundred F2 mice (fifty males and fifty females per group) were
   randomly assigned to each of nine exposure groups for a total of 900
   mice in the study population. When assigning mice to each group, only
   one same sex animal from any given litter was assigned to the same
   treatment to eliminate litter effects. Study mice were weaned at
   post-natal day 21 onto AIN-93M standard diet (Envigo-Teklad Diets). At
   6-weeks mice were switched from AIN-93M diet onto an American-style
   diet (Envigo-Teklad Diets) that was designed to be similar to the
   typical American diet with increased kcal from fat, a skewed omega 6 to
   omega 3 fatty acid ratio, and deficient for folic acid compared to the
   AIN-93M standard diet. Mice were allowed to acclimate to the American
   diet for 10-14 days before being assigned to a specific exposure group.
   Entry dates into specific treatment cohorts were staggered to minimize
   confounding by calendar date of procedures, with 200-300 F3 mice
   entering the study every four to five weeks over a 16-week period.
   Exposure groups were designed to include no, low, and high doses of
   each toxicant and all possible combinations of these categories
   ([61]Table 1). Exposure dosages were calculated as dose equivalents
   based on human exposure data and low and high doses were selected to be
   no more than 2 to sixfold different than actual human exposures. For
   TCE these values included well water measurements of TCE for the high
   dose and data from the National Health and Nutrition Examination Study
   (NHANES) for the low dose ([62]National Research Council (U.S.)
   Committee on Human Health Risks of Trichloroethylene. 2006). Doses for
   arsenic were based on the World Health Organization (WHO) limit for the
   low dose and a calculation of arsenic in rice combined with average
   daily intake for the high dose ([63]Stone 2008). TCE (0, 5, or 2850
   ppb) was added to purified drinking water, prepared fresh weekly, and
   administered in UV-light protected bottles to prevent degradation. iAs
   (0, 10, or 150 μg/kg as sodium arsenite) was mixed into the
   cAmerican-style diet. All food was replaced weekly and stored at 4° in
   vacuum-sealed bags to prevent oxidation. Mice were maintained on their
   assigned treatment group for the 52-week duration of the toxicant
   exposure. All sample collections and measurements were performed during
   narrow time windows to minimize circadian effects.

Table 1. Treatment groups.

   Group Dose Ratio (iAs:TCE) iAs Concentration (μg/kg food) TCE
   Concentration (ppb in water)
   1 None: None 0 0
   2 None: Low 0 5
   3 None: High 0 2850
   4 Low: None 10 0
   5 Low: Low 10 5
   6 Low: High 10 2850
   7 High: None 150 0
   8 High: Low 150 5
   9 High: High 150 2850
   [64]Open in a new tab

Sample Collection

   Urine was collected prior to the beginning of toxicant exposure, at
   intermediate times after 19 weeks and 32 weeks of exposure, and prior
   to euthanasia following 52 weeks of exposure. Mice were individually
   housed in diuresis cages (Hatteras Instruments Inc., Cary NC) for 16 hr
   with ad libitum access to drinking water. Urine collection tubes were
   maintained at 1° to 6° for the duration of collection. Urine was
   centrifuged at 10,000 × g for 5 min to separate particulate matter, 30
   μL aliquots were made into fresh clear polypropylene tubes, and
   aliquots and original sample tube were stored at -80° for future
   analysis. Samples from the collection just prior to termination were
   analyzed for this study.

   Blood was collected at the same time points as urine. Briefly, blood
   was collected from the mandibular vein using capillary tubes for
   collections done prior to and during toxicant exposure. Terminal blood
   collection was performed by cardiac puncture at the time of necropsy.
   Blood was allowed to clot for up to 30 min at room temperature in
   tubes. The blood was then centrifuged at 10,000 × g for 10 min to
   separate serum from erythrocytes and leukocytes. Serum was transferred
   into fresh 1.5 mL clear Eppendorf polypropylene tubes and stored at
   -80° for future analysis. Serum from the terminal collection was
   analyzed for this study.

   Following 52 weeks of toxicant exposure, mice were killed by carbon
   dioxide asphyxiation followed by cervical dislocation. At necropsy
   kidneys and other organs (liver, lung, heart, and any organs with
   abnormal presentations) were removed, weighed, and examined for gross
   abnormalities. Organ samples were fixed for histology or flash frozen
   for molecular analysis.

Histopathologic Examination

   Each kidney was halved longitudinally. One half of each kidney was
   fixed in 10% neutral buffered formalin for 24 hr, transferred to 70%
   ethanol, then routinely processed and paraffin embedded. Five 5μm-thick
   serial sections were obtained and the first, third and fifth of these
   were hematoxylin and eosin (H&E) stained for histopathological
   examination. Light microscopic examination of kidney slides was
   performed by a board-certified anatomic pathologist (AP). Each slide
   was randomly assigned a new identifier to mask exposure group from the
   pathologist. The first, third and fifth slides for each individual were
   examined for neoplasia or preneoplastic changes. Following this initial
   examination, one representative slide from each individual was examined
   and scored for histologic evidence of renal disease. Only after all
   samples were scored was the code broken to determine which samples were
   from each treatment group.

Measurement of BUN and Creatinine

   Measurement of serum blood urea nitrogen (BUN) and creatinine was
   performed in house using a VetScan VS2 Chemistry Analyzer (Abaxis,
   Union City, CA) with VetScan Comprehensive Diagnostic Profile Rotors.
   Serum samples were thawed on ice, mixed with an equal volume of 0.9%
   saline solution to obtain a final volume of 100 μL, pipetted into the
   rotor, and analyzed. Creatinine concentrations were below the limits of
   detection for this instrument for the majority of animals and could not
   be analyzed.

Measurement of Urine Protein and Creatinine

   Urine protein quantification was performed using a Pierce Coomassie
   Plus Bradford Assay (Thermo Fisher). A urine sample aliquot was thawed
   on ice and the assay was performed per manufacturer’s protocol.
   Briefly, urine samples were diluted with MilliQ water (1:500 for
   females, 1:1000 for males) and pipetted into microplates. The Coomassie
   reagent was brought to room temperature before adding to all wells of
   the plate and incubating at room temperature for 10 min. Samples were
   measured in duplicate using a microplate reader at 595 nm. Urine
   protein concentration (mg/dL) was determined based on a standard curve.

   Urine creatinine measurement was performed using a Creatinine (urinary)
   Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, MI). A urine sample
   aliquot was thawed on ice and the assay was performed according to the
   manufacturer’s protocol. Briefly, urine samples were diluted with
   MilliQ water and added in duplicate to the wells of a microplate.
   Alkaline picrate solution was added to all wells and incubated on a
   shaker for 10 min at room temperature. Initial absorbance was read at
   500 nm. Acid solution was added to all wells and the plate was
   incubated for an additional 20 min at room temperature before the final
   absorbance measurement. Samples were measured in duplicate using a
   microplate reader at 500 nm. Urine creatinine concentration (mg/dL) was
   determined based on a standard curve.

Measurement of Urinary NGAL

   Urinary Neutrophil Gelatinase Associated Lipocalin (NGAL) measurement
   was performed using a Mouse Lipocalin-2/NGAL Quantikine ELISA kit (R&D
   Systems). Urine aliquots were thawed on ice, diluted in the provided
   assay buffer, and the assay was performed according to the
   manufacturer’s protocol. Briefly, assay buffer was added to all wells
   of the kit-provided microplate, samples and standards were added in
   duplicate, followed by incubation at room temperature. The plate was
   aspirated and washed, followed by conjugate addition and incubation,
   substrate incubation in light-protected conditions, and addition of
   stop buffer. Samples were measured in duplicate using a microplate
   reader at 540nm/450nm within 30 min of adding the stop buffer. Urine
   NGAL concentration was determined based on a standard curve. Urine NGAL
   concentrations were then normalized to urine creatinine and urine
   osmolality measurements to account for variability in sample
   concentration.

Gene Expression Analysis

   Frozen kidney samples from low single exposures and low combination
   exposure groups were thawed and total RNA extracted with an RNAeasy kit
   (Qiagen). RNA samples were sent to the University of North Carolina
   Functional Genomics Core for global gene expression analysis with
   GeneChip Mouse Gene 1.1 ST arrays (Affymetrix, Santa Clara). Gene
   expression data (CEL files) were analyzed using Partek Genomic Suite, v
   6.5 (Partek, St. Louis). Data were normalized by robust multiarray
   analysis and analysis of variance (ANOVA). Differentially expressed
   genes were identified between classes using the non-parametric
   rank-product method (P < 0.05), and log[2] expression values for each
   gene were generated. Canonical pathway analysis was generated through
   Ingenuity Pathway Analysis (Qiagen). Functional classes were compared
   to a previous report ([65]Desimone et al. 2013).

Statistical Analyses

   Statistical analysis of Kaplan-Meirer survival curves was performed
   using a log-rank test in Mstat ([66]Drinkwater 2018). Statistical
   analyses of histopathologic scoring were performed using JMP 13.0 (SAS,
   Raleigh, NC) and graphs were built in Prism 8 (GraphPad, San Diego,
   CA). Tubular disease scores were analyzed by ANOVA with post-hoc
   testing using the Dunn method for multiple comparisons which utilizes
   the Bonferroni adjustment to correct for multiple comparisons. The
   control group for statistical analyses was defined as the untreated
   group. Biomarkers including BUN, urine protein/creatinine ratio, and
   urinary NGAL were log transformed to approximate normality, then
   analyzed by ANOVA with post-hoc testing using the Dunnet method for
   multiple comparisons with the control group defined as the untreated
   group.

Data Availability

   All primary data are available in the supplemental file or upon
   request. Supplemental material available at Figshare:
   [67]https://doi.org/10.25387/g3.7871525.

Results

Survival Is Affected Only by High iAs Exposures

   No significant differences were found in a Kaplan-Meier survival
   analysis through the end of the study between any dose group lacking
   High iAs treatment and the No iAs/No TCE (control) group ([68]Figure
   1). Log-rank testing of survival showed a significant difference among
   the groups (P < 0.001). Subsequent multiple comparisons showed only the
   High iAs/Low TCE (P = 0.004) and High iAs/High TCE (P < 0.001) having
   higher mortality than the control group, with only the High iAs/High
   TCE having higher mortality than all other treatment groups except High
   iAs/Low TCE.

Figure 1.

   [69]Figure 1
   [70]Open in a new tab

   Kaplan-Meier survival plots by dose group.

Toxicant Exposure Is Associated With Renal Tubular Disease

   While no changes in overall survival to the end of the study were
   observed, significant differences between dose groups in tubular
   disease score as measured by histologic examination were observed.
   Lesions included in the evaluation were based on numerical criteria
   scoring ([71]Table 2; [72]Figure 2). Diagnoses were based on criteria
   published as part of the International Harmonization of Nomenclature
   and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) Project
   ([73]Frazier et al. 2012). Tubular disease lesion scores were
   determined by summing the individual scores in each category resulting
   in a total score for each animal between 0 and 20 (raw scores are in
   Supplemental Table S1).

Table 2. Renal damage scoring matrix.

   Score Chronic progressive nephropathy (CPN) Tubular degeneration +/−
   regeneration Tubular dilation Karyomegaly Tubular single cell necrosis
   Tubular epithelial microvesicles
   0 None None None None Absent None
   1 Rare affected tubules Rare tubules with evidence of degeneration or
   regeneration not associated with lesions of CPN Rare tubules with
   lumens ≥50% of total tubule diameter Rare tubular cells with nuclei ≥2X
   the size of normal nuclei Present Present in rare tubules
   2 Few foci of affected tubules Few tubules with evidence of
   degeneration or regeneration not associated with lesions of CPN Few
   tubules with lumens ≥50% of total tubule diameter Few tubular cells
   with nuclei ≥2X the size of normal nuclei N/A Present in few tubules
   3 Many foci of affected tubules Many tubules with evidence of
   degeneration or regeneration not associated with lesions of CPN Many
   tubules with lumens ≥50% of total tubule diameter Many tubular cells
   with nuclei ≥2X the size of normal nuclei N/A Present in many tubules
   4 Most tubules affected Most tubules have evidence of degeneration or
   regeneration not associated with lesions of CPN Most tubules have
   lumens ≥50% of total tubule diameter N/A N/A Present in most tubules
   [74]Open in a new tab

Figure 2.

   [75]Figure 2
   [76]Open in a new tab

   Representative examples of renal pathologies and non-renal tumors. (A)
   Male. High As/High TCE exposure group. Tubular dilation (asterisk),
   degeneration and regeneration. One tubule has a necrotic tubular
   epithelial cell (arrow). (B) Male. No As/Low TCE exposure group.
   Chronic progressive nephropathy lesions. There are areas of relatively
   well demarcated tubular change consisting of basophilic tubules with
   thickened basement membranes. A glomerulus in the section is expanded
   by presumed amyloidosis. (C) Female. Low As/High TCE exposure group.
   Lymphoma. (D) Female. High As/High TCE exposure group. Histiocytic
   sarcoma. Hyaline droplets in renal tubular epithelium support the
   diagnosis (inset, lower right).

   Comparisons were made between each treatment group and the unexposed
   group. Increases in mean tubular disease scores were detected in the No
   iAs/Low TCE (mean score = 3.47, P < 0.0001), No iAs/High TCE (mean
   score = 2.83, P = 0.0184), Low iAs/No TCE (mean score = 3.76, P <
   0.0001), Low iAs/High TCE (mean score = 3.53, P < 0.0001), High iAs/No
   TCE (mean score = 4.00, P < 0.0001), and High iAs/High TCE (mean score
   = 3.48, P < 0.0068) groups as compared to the No iAs/No TCE group (mean
   score = 1.53) ([77]Figure 3A). In addition, increases in tubular
   disease scores were observed in animals exposed to TCE alone, iAs
   alone, and in those exposed to both toxicants in combination, although
   there was no increase in average severity of disease in those with
   combination exposure to TCE and iAs compared to those with single
   toxicant exposure ([78]Figure 3B).

Figure 3.

   [79]Figure 3
   [80]Open in a new tab

   Renal disease scores. (A) Mean (+SE) tubular disease score in each dose
   group. Number of mice analyzed from each group is shown above. No As/No
   TCE group was used as control for comparisons: **** P < 0.0001; ** P =
   0.0068; * P = 0.0184. (B) Mean (+SE) tubular disease score by toxicant
   exposure or co-exposure. Number of mice in each group is noted at the
   bottom. No Toxicant group used as control for comparisons: **** P <
   0.0001, *** P = 0.0003.

Males Have Greater Histological Evidence of Renal Disease

   Further analysis of the histological tubular disease scoring was
   performed to investigate the effects of TCE dose, iAs dose, and sex on
   renal tubular damage. After determining that data were normally
   distributed, ANOVA analysis showed that sex (P = 0.0006) was a
   significant factor, and that there was a significant interaction
   between TCE dose and iAs dose (P = 0.0005). A Welch’s t-test was
   performed, and, overall, male mice had higher mean total tubular
   disease scores (mean score 3.678) than female mice (mean score 2.683, P
   < 0.0001).

   In addition, male mice in general had higher log transformed mean
   scores for nearly all of the assessed individual renal lesions on
   histologic examination including: glomerular amyloid/hyaline
   glomerulopathy (P < 0.0001), medullary amyloid or fibrosis (P <
   0.0001), end-stage kidney (P = 0.0034), perivascular cellular
   infiltrate (P = 0.005), chronic interstitial cellular infiltrate (P =
   0.028), tubular degeneration and regeneration (P = 0.0134), tubular
   single cell necrosis (P = 0.0006), pelvic dilation (P < 0.0001),
   infarcts (P < 0.0001), and hyaline casts (P < 0.0001). This pattern of
   greater histologic evidence of renal damage held for the biomarkers
   assessed as well, with UPC, BUN, and urinary NGAL normalized against
   creatinine all higher in male mice than in female ([81]Figure 4).

Figure 4.

   [82]Figure 4
   [83]Open in a new tab

   Renal parameters separated by sex. (A) Mean (+SE) tubular damage across
   all dose groups. Male n = 171; female n = 278 (P < 0.0001). (B) Mean
   (+SE) urine protein/creatinine ratio across all dose groups. Male n =
   113, female n = 233 (P < 0.0001). (C) Mean (+SE) BUN across all dose
   groups. Male n = 156, female n = 264 (P < 0.0001). (D) Mean (+SE) NGAL
   normalized to creatinine across all dose groups. Male n = 113, female n
   = 233 (P = 0.0027).

Combination Exposure Does Not Increase Histologic Evidence of Tubular Damage

   The highest mean damage score resulted from high iAs and no TCE
   exposure, but no significant difference was observed between the high
   iAs/high TCE condition and the high iAs/low TCE condition ([84]Figure
   3A). As expected, lowest damage occurred with no toxicants.
   Interestingly, when the TCE level is high, no significant effects were
   seen with the addition of iAs exposure. When TCE level is low, higher
   damage was observed without iAs, and lower damage scores were found
   when low or high iAs was included. When individual kidney lesion types
   were examined, those that were more common in toxicant-exposed animals,
   whether single or combination exposure, compared to the No iAs/No TCE
   control group were: pelvic cellular infiltrates (P = 0.0005), tubular
   degeneration and regeneration (P < 0.0001), tubular dilation in the
   outer stripe of the outer medulla (OSOM) (P = 0.0022), pelvic dilation
   (P = 0.0254), and epithelial vacuolation of tubular epithelium (P =
   0.0168). Among these lesions, pelvic cellular infiltrates were higher
   in animals exposed to each single toxicant (TCE only P = 0.0064; iAs
   only P = 0.0033) and combination-exposed animals (P = 0.0274) as
   compared to the unexposed group, but there was no difference between
   combination-exposed animals and single-exposed animals. This pattern
   held for tubular degeneration and regeneration (TCE only P < 0.0001; As
   only P < 0.0001; combination exposure P = 0.0011), except that arsenic
   only exposure resulted in higher tubular degeneration and regeneration
   score compared to combination-exposed animals (P = 0.0004). For tubular
   dilation of the OSOM, both single exposure to TCE (P = 0.0386) and
   single exposure to iAs (P = 0.0003) resulted in higher scores for this
   parameter compared to those not exposed, but single exposure to iAs
   resulted in higher scores for this parameter compared to combination
   exposure (P = 0.0100). For tubular vacuolation, only combination
   exposure resulted in higher scores for this parameter and only in
   comparison to unexposed animals (P = 0.0296).

BUN Level Correlates With Histology Parameters

   Blood urea nitrogen concentrations across the study population were
   analyzed for correlation to the tubular disease score as well as the
   urinalysis parameters. BUN values were log transformed before Pearson’s
   correlation testing was performed. Moderate positive linear
   correlations were observed between BUN and kidney weight/body weight
   ratio (0.4268), NGAL normalized to creatinine (0.4349), and NGAL
   normalized to osmolality (0.3687) (P < 0.0001 for all). A slight to
   moderate positive correlation was observed between BUN and urine
   protein/creatine (UPC) ratio (0.3237). However, the strongest
   correlation was found between BUN and tubular disease score (0.4657).

   BUN concentrations for single and combination exposed groups were
   compared to the No iAs/No TCE group after log transformation of the BUN
   values to improve normality. ANOVA followed by the post-hoc
   Tukey-Kramer HSD test indicated that BUN was higher in single iAs
   exposed animals compared to unexposed animals, but no other differenced
   were observed. BUN levels for each exposure group were also analyzed
   and compared to the No iAs/No TCE group using ANOVA with post hoc
   testing using Dunnett’s method. Significance was reached only in the
   Low iAs/No TCE group (P = 0.0137) and the High iAs/No TCE group (P =
   0.0333) when compared to the No iAs/No TCE group used as the control
   for this analysis ([85]Figure 5A).

Figure 5.

   [86]Figure 5
   [87]Open in a new tab

   Clinical biomarkers of kidney injury. (A) BUN levels for each exposure
   group were analyzed and compared to the No iAs/No TCE group.
   Significance was reached only in the Low iAs/No TCE group (P = 0.0137)
   and the High iAs/No TCE group (P = 0.0333) when compared to the No
   iAs/No TCE group used as the control for this analysis. (B) Urine
   protein/carnitine ratios for male and female mice across the entire
   study population. Male n = 113, female n = 233 (P < 0.0001). (C) Urine
   protein/carnitine ratios for male and female mice in the No As/No TCE
   group. Male n = 12, female n = 31 (P < 0.0001).

   Urinary NGAL was measured and normalized against urinary creatinine.
   Despite its reported association with tubular damage and slight to
   moderate correlation with the tubular disease score obtained from
   histologic examination of the kidneys (0.3395 for NGAL normalized
   against creatinine and 0.2934 for NGAL normalized against osmolality),
   no difference was observed in urine NGAL normalized against creatinine
   (P = 0.4443) among the exposure groups, or between single or
   combination exposed animals compared to the No iAs/No TCE group.

   When UPC ratios were compared between the toxicant-exposure groups and
   the No iAs/No TCE group used as a control, the UPC ratio was higher
   only in the Low iAs/High TCE group (P = 0.0082) and the High iAs/No TCE
   group (P = 0.0145). Male mice across the entire study population had
   higher UPC values (P < 0.0001) ([88]Figure 5B), which was expected
   given that male mice are known to have higher levels of urinary
   proteins than female mice. When UPC levels for the entire study
   population were compared to the UPC levels in the no toxicant control
   group, the magnitude of the increase was higher among toxicant-exposed
   mice than in the control population ([89]Figure 5C).

Exposure and Sex Influence of Non-Renal Neoplasias

   No tubular epithelial neoplasms or pre-neoplastic changes of tubular
   epithelium were observed in the examined sections from any group.
   However, eight instances of infiltrative round cell neoplasms
   (histiocytic sarcoma and presumed lymphoma) affecting the kidney were
   identified ([90]Table 3; [91]Figure 2). Of the eight round cell
   neoplasms identified in the kidneys, seven were in female mice, and
   four of the eight were identified in mice with iAs exposure but no TCE
   exposure.

Table 3. Tumor observed in mice surviving to 52-weeks.

   Dose Group Sex Tumor Type Other Affected Organs
   No iAs/No TCE F Histiocytic sarcoma None detected
   No iAs/Low TCE F Lymphoma None detected
   Low iAs/No TCE F Histiocytic sarcoma Liver, Lung
   Low iiAs/No TCE F Histiocytic sarcoma Liver
   Low As/No TCE F Lymphoma Liver, Lung
   Low iAs/High TCE F Lymphoma Liver
   High iAs/No TCE M Lymphoma Liver, Lung, Cranial mediastinal mass
   High iAs/High TCE F Histiocytic sarcoma Lung, Lymph node, Cranial
   mediastinal mass
   [92]Open in a new tab

Combination Exposure Causes Molecular Changes Similar to That of a
TCE-Associated VHL Mutation From Renal Cell Carcinomas

   Kidney samples from mice that survived 52-weeks of exposure were
   randomly selected for genome-wide gene expression analysis on
   Affymetrix Gene 1.1 ST arrays. Hierarchical clustering of all genes
   that were differentially expressed among the samples revealed that the
   samples clustered tightly by treatment ([93]Figure 6A). Genes that were
   differentially expressed between the Low iAs/ Low TCE combination and
   the two individual treatment groups were over-represented in genes
   associated with two tumor suppressor genes (Tsc2 and Vhl) known to be
   involved with human renal cell carcinoma ([94]Figure 6B). Furthermore,
   pathway enrichment analysis of the Low iAs/Low TCE differentially
   expressed genes identified the same pathways previously reported for
   the VHL P81S mutation from renal cell carcinomas in people exposed to
   industrial levels of TCE ([95]Desimone et al. 2013). These pathways
   included those in renal cell carcinoma signaling, anti-apoptotic
   response, hypoxia signaling, and cellular metabolism.

Figure 6.

   [96]Figure 6
   [97]Open in a new tab

   Molecular changes in kidney from exposed mice. (A) Hierarchical
   clustering of differentially expressed genes. (B) Ingenuity Pathway
   Analysis of genes differentially expressed in Low iAs/Low TCE exposure
   groups. (C) Pathway enrichment analysis of genes differentially
   expressed in Low iAs/Low TCE exposure groups.

Discussion

   Developing an accurate rodent model for human TCE-associated renal cell
   carcinoma has been challenging. Toxicological studies generally
   evaluate toxicants in isolation and in genetically homogeneous
   populations of rodents, even though this does not reflect the genetic
   variability of exposed human populations or the typical scenario of
   exposure in which multiple toxicants are often encountered together.
   Response to toxicant exposure is governed by many factors including
   intrinsic (genetic and epigenetic variation, age and life stage, sex)
   and extrinsic factors (co-exposures to other toxicants, nutritional
   state, stressors, dosage, co-morbidities) ([98]Zeise et al. 2013).

   In this study, we analyzed long-term exposure to TCE alone or in
   combination with iAs. Previous studies have shown large variability
   among strains in metabolism and response to TCE ([99]Cichocki et al.
   2017), suggesting long-term TCE exposure in genetically heterogeneous
   mice could lead to renal cell carcinoma. In addition to including
   genetic heterogeneity and nutrition modeled on the typical American
   diet, our study included a second common environmental toxicant, iAs,
   to investigate the potential interactions of two commonly occurring
   toxicants. Arsenic is a known renal toxicant and urinary system
   carcinogen when exposure occurs through drinking water
   ([100]Christoforidou et al. 2013), and is also known to accumulate and
   become concentrated in plant-based food products grown in
   arsenic-containing water ([101]Davis et al. 2012; [102]Gilbert-Diamond
   et al. 2011; [103]Jackson et al. 2012).

   Surprisingly, little differences were observed in survival among dose
   groups except those on High iAs and any dose of TCE. Even the control
   group had a relatively large mortality over the 52-week study period,
   which was almost three-times higher in males than females. One factor
   contributing to lack of survival differences was conspecific
   aggression, relevant to control and exposed groups. Aggression among
   laboratory mice, particularly male mice, is a known issue and has
   proven difficult to reduce in group housing. Many variables are
   involved in aggression between laboratory mice, including but not
   limited to size of housing, number of animals per cage, bedding,
   shelters, temperature, strain, and stress ([104]Weber et al. 2017).
   Efforts were made in the present study to limit the effects of
   aggression, such as the use of larger than standard cages and limiting
   exposure to unfamiliar animals. Even so, some actions that may limit
   aggression, for example keeping littermates together, could not be
   implemented in the present study in favor of limiting litter effects.

   Although no renal cell carcinomas were observed, significant
   differences in histologic and biomarker evidence of renal tubular
   disease were observed among the different treatment groups, including
   increases in tubular disease scores between animals exposed to TCE
   alone, exposed to iAs alone, and those exposed to both toxicants,
   although no difference in severity was observed in those co-exposed,
   contrary to our expectations. It is possible that damage caused by one
   toxicant was not increased by exposure to a second renal toxicant as
   the cells already damaged by the first toxicant, to the point of
   requiring regeneration, would not be further damaged by the second
   toxicant. The study was designed to increase renal carcinogenic
   potential by looking a more diverse genetic backgrounds and adding a
   second common toxicant. The lack of renal cell carcinomas suggests that
   these were not limiting factors. Even though renal cell carcinomas were
   not observed, molecular analysis of kidneys from mice exposed for
   52-weeks showed unique gene expression changes associated with combined
   Low iAs/Low TCE exposure. The changes indicated a kidney cells
   manifested a tumor promotion state only from the combined exposure that
   was similar to gene expression changes elicited in an embryonic stem
   cell assay for the renal cell carcinoma mutation, VHL P81S, observed in
   patients exposed to TCE in an industrial setting ([105]Desimone et al.
   2013). These results support the link between TCE exposure and
   molecular changes leading to renal cell carcinoma. Only recently have
   mice been induced to develop renal cell carcinoma by combining
   mutations in Vhl, Trp53, and Rb1 ([106]Harlander et al. 2017). Thus,
   the reason renal cells in the exposed mice did not progress to cancer
   is likely due to the fact that Vhl mutations alone are not sufficient
   in to induce renal cell carcinoma in mice.

   Further limitations are that the rate of renal cell carcinoma in humans
   is relatively low and thus the incidence in an animal model might be
   expected to reflect this. The annual incidence in the United States is
   15.6 per 100,000 people (National Cancer Institute). Even in higher
   incidence areas, such as Camp LeJeune, a TCE-contaminated Superfund
   site, the incidence rate is only 21.3 per 100,000 people (Centers for
   Disease Control and Prevention), making the study of these tumors
   difficult even in a rodent model, due to the large number of
   individuals required to detect the formation of these tumors. Another
   factor complicating the modeling of this cancer in rodents is that it
   requires long periods of time for tumors to develop, increasing the
   incidence of age-associated sporadic lesions, such as chronic
   progressive nephropathy (CPN), that complicate histopathologic
   interpretation. Further, rodents, especially mice, have a higher
   capacity for metabolism of TCE than humans, ([107]Lash et al. 2000),
   making the toxic metabolite profile and subsequent organ system
   pathology potentially differ from that seen in humans.

   Despite these limitations, the current study used a model more
   accurately reflecting human exposure conditions by including multiple
   toxicants, environmentally relevant concentrations, a genetically
   heterogeneous population, and a long period of exposure. Although a
   more accurate model of human exposure conditions was used, the primary
   renal tumor development observed in humans following chronic TCE
   exposure was not observed in this study. Renal tubular disease did
   occur both in all treatment groups, and a unique gene expression
   signature similar to that elicited by expressing a TCE-associated VHL
   mutation from renal cell carcinomas was observed specifically in the
   iAs and TCE combined treatment group.

Acknowledgments