Female Rat Behavior Effects from Low Levels of Hexavalent Chromium (Cr[VI]) in Drinking Water Evaluated with a Toxic Aging Coin Approach

Abstract

We are facing a critical aging crisis, with geriatric populations (65+) growing to unprecedented proportions and ~4 million people (a 6.5-fold increase) expected to become centenarians by 2050. This is compounded by environmental pollution, which affects individuals of all ages and contributes to age-related diseases. As we have a limited understanding of how environmental pollutants affect older populations distinctly from younger populations, these longer-lived geriatric populations present a key knowledge gap. To address this knowledge gap, we employ a “Toxic Aging Coin” approach: heads consider how age impacts chemical toxicity, and tails consider how chemicals act as gerontogens—or how they accelerate biological aging. We employed this approach to investigate hexavalent chromium (Cr[VI]) impacts on female rats exposed to 0.05 or 0.1 mg Cr(VI)/L in drinking water for 90 days; these are the maximum contaminant levels (i.e., the highest levels permitted) from the World Health Organization and U.S. Environmental Protection Agency, respectively. During exposure, rats performed a battery of behavior assays to assess grip strength, locomotor coordination, anxiety, spatial memory, sociability, and social novelty preference. We observed age differences in Cr(VI) neurotoxicity, with grip strength, locomotor function, and spatial memory in middle-aged females being particularly affected. We further compared these results in females to results in males, noting many sex differences, especially in middle-aged rats. These data emphasize the need to consider age and sex as variables in toxicology and to revisit drinking water regulations for Cr(VI).

1. Introduction

We are facing an unprecedented aging crisis, arising from a combination of the baby boomer generation reaching a geriatric age and the human lifespan increasing by approximately 2 years every decade [1,2]. In the next 25 years, 20% of the global population will be 65+ years old, and approximately 3.6 million people (a 6.5-fold increase) will be 100+ years old [1,3]. These longer-lived populations portend an increased prevalence of age-related diseases and comorbidities. Compounding this aging crisis, environmental pollution is a persistent threat to human health, and older populations are often more vulnerable to the negative health effects of environmental pollutants [4]. Historically, geriatric populations have been largely omitted from environmental toxicology studies due to their proximity to death, as it typically takes multiple decades of exposure to result in diseases. Now, our geriatric populations are predicted to live for several decades, and we have limited knowledge of how long-term chemical exposures affect geriatric populations distinctly from younger populations.

Environmental pollution interacts with the aging crisis in two critical ways: (1) geriatric populations are often more vulnerable to chemical health threats, and (2) environmental pollutants may act as gerontogens—a class of chemicals that induce or accelerate aging processes [5,6]. Evidence suggests geriatric individuals are often more vulnerable to the negative health effects of environmental pollution, but age as a biological variable is not a major consideration in toxicology [5]. Many chemicals act as gerontogens, and this number is increasing as more pollutants are considered [6,7,8,9,10]. To address the interactions between aging and toxicology, we employed a “Toxic Aging Coin” approach [10]. On the heads side, we consider how age impacts toxicity, while on the tails side, we consider how chemicals act as gerontogens.

Metals are particularly hazardous environmental pollutants, as they are naturally occurring, often in industrial waste and effluent. Many are toxic, and remediation is difficult, as most cannot be metabolized or degraded to less toxic forms. Metals are considered a class of gerontogens, as exposures are linked to all the top 10 most prevalent age-related diseases, and many metals induce hallmarks of aging across multiple tissues (e.g., cellular senescence, telomere attrition, mitochondrial dysfunction) [6,11,12,13,14,15,16]. Chromium is a naturally occurring metal found in the environment in two valence states—trivalent (Cr[III]) and hexavalent (Cr[VI]). Cr(III) is relatively innocuous to human health, while Cr(VI) is regarded as highly toxic and a known human carcinogen [17,18]. Many reports have shown that Cr(VI) levels in drinking water exceed the maximum contaminant levels set by the World Health Organization (WHO) and the U.S. Environmental Protection Agency (US EPA) at 0.05 and 0.1 mg/L, respectively. Such water contamination is often due to industrial pollution and sometimes due to geogenic sources from ultramafic volcanic bedrock [19,20,21,22,23]. Cr(VI) exposure may be a risk factor for at least 3 of the top 10 age-related diseases: chronic obstructive pulmonary disease, Alzheimer’s disease, and chronic kidney disease [24,25,26]. Further, evidence from welders occupationally exposed to Cr(VI) in welding fumes and cell culture studies suggests Cr(VI) acts as a gerontogen [27,28,29,30,31].

Decades of research have reported Cr(VI) neurotoxicity in cell cultures, animal models (rodents, fish, birds, Drosophila), and human populations, but many key knowledge gaps remain [10]. In humans, Cr(VI) neurotoxicity can differ across age groups. Childhood exposure is linked to autism spectrum disorder or attention deficit hyperactivity disorder, whereas adult exposure is linked to olfactory dysfunction, memory loss, polyneuropathy, and motor neuron disease [10,32,33,34,35,36,37,38]. Cr is reported to accumulate in the brains of non-occupationally exposed individuals, with the highest levels reported in the temporal cortex and pituitary gland [39]. Notably, older individuals exhibited higher Cr levels in brain tissues, suggesting Cr accumulates in the brain with age or that geriatric individuals accumulate more Cr in the brain upon exposure [39]. Animal studies report Cr(VI) neurotoxicity following injection, inhalation, or drinking water exposure and across multiple taxa (fish, birds, mammals, Drosophila) [39]. However, previous drinking water studies used unrealistic levels that exceeded the maximum contaminant limits by at least 700× [39,40]. Regardless of the animal model or exposure route, Cr(VI) neurotoxicity studies repeatedly report impaired locomotor function, decreased activity, learning and memory deficits, widespread neurodegeneration, and widespread oxidative damage [39,40,41,42,43]. However, behavior assessments have been limited in scope, and there is little consideration for age or sex as biological variables.

To address these knowledge gaps, we considered Cr(VI) neurotoxicity in Sprague-Dawley rats across three distinct ages (3, 7, and 18 months old) exposed to low concentrations of Cr(VI) (0.05 and 0.1 mg/L) (Figure 1). Critically, these drinking water levels reflect the maximum contaminant levels from the World Health Organization (WHO) and the U.S. Environmental Protection Agency (US EPA). Here, we present data from female rats and report differences compared to previously published results in male rats [44]. During exposure, rats performed a battery of behavioral assays to assess Cr(VI) effects on grip strength, locomotor function, anxiety, spatial memory, activity, and social memory (

Table 1. Schedule of Behavior Assays Over the 90-Day (13 Weeks) Exposure Period.

Week #	Behavior Assay	Behavior(s) Assessed
0	(Cr[VI] Exposures Started)
1	Grip Strength Test	Neuromuscular function
2	Open-Field Assay	Exploration, rearing, locomotion
3	Elevated Plus Maze	Anxiety
4	Y-Maze	Spatial memory, activity
5	3-Chamber Assay	Sociability, social novelty preference
6	(No behaviors assessed)
7	Grip Strength Test	Neuromuscular function
8	Open-Field Assay	Exploration, rearing, locomotion
9	Elevated Plus Maze	Anxiety
10	Y-Maze	Spatial memory, activity
11	3-Chamber Assay	Sociability, social novelty preference
12	(No behaviors assessed)
13	Rats Sacrificed

).

2. Materials and Methods

2.1. Animals

Rats were maintained in facilities fully accredited by AAALC International, and all experiments were performed under the protocol IACUC 21934 approved by the University of Louisville Institutional Animal Care and Use Committee in accordance with the ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study design for female rats is shown in Figure 1. Data presented here included 81 female Sprague-Dawley rats purchased from Envigo (Indianapolis, IN, USA) at 3, 7, or 18 months old and enrolled in the study within 10 days of arriving at UofL facilities. Upon termination of the study, after 13 weeks of Cr(VI) exposure, groups were 6-, 10-, or 21-months old. Rats were fed a rodent diet with 10% kcal from fat (Research Diets, New Brunswick, NJ, USA, D12450Ji). Rats from each age were divided into 3 exposure groups, for a total of 9 groups: study groups received tap water, 0.05 mg Cr(VI)/L in tap water, or 0.1 mg Cr(VI)/L in tap water, reflecting maximum contaminant levels set by WHO and US EPA, respectively. The young and middle-aged groups included 8 rats per group, while the geriatric groups included 9 or 12 rats for the control and Cr(VI)-treated groups, respectively, to account for attrition due to age. In total, three female rats were lost to attrition: a control geriatric rat died due to natural causes prior to the beginning of Cr(VI) exposure, and during weeks 10 and 11 of the study, a geriatric rat exposed to 0.05 mg Cr(VI)/L and a control geriatric rat were sacrificed early due to health complications. The group sample size (n = 8) enabled us to detect an effect size (i.e., the ratio of group mean difference to within-group standard deviation) of 1.8, with a power of 80% at a significance level of 0.05.

Cr(VI) was administered as sodium chromate (Sigma Aldrich, St. Louis, MO, USA 307831) dissolved in tap water. Cr(VI) exposures were prepared weekly from 1000× stocks in tap water: 50 mg Cr(VI)/L and 100 mg Cr(VI)/L stocks were diluted to 0.05 and 0.1 mg Cr(VI)/L final concentrations in tap water. Body mass and drinking water mass were measured weekly, and no differences were observed across the exposure groups. For QA/QC, drinking water samples were collected weekly for metal analyses to validate the Cr(VI) concentration.

2.2. Behavior Assays

Various chambers for open-field assay, elevated plus maze, Y-maze, and 3-chamber assay were built by Dr. Jun Cai. Rats performed one assay per week (Table 1), and 70% ethanol was used to sanitize and deodorize the equipment between each rat.

All behavior assays were performed in the same room, under red light, and enclosed with foam boards to reduce ambient noise and obstruct the test rat’s view of the experimenter. All behavior assays were performed in the afternoons during the light period of the 12-h light-dark cycle to reduce variability from the circadian rhythm. Due to the large number of animals in this study, behaviors were only able to be assessed twice and were designed to be equal weeks apart. There was no scientific or biological rationale for the sequence of behavior assays; rather, it was based on the availability of the equipment and facility.

We recorded aerial and side views of behavior assays using a 4 mm C Series Fixed Focal Length Lens camera (Edmund Optics, Barrington, NJ, USA, 33300) and a 6 mm C Series Fixed Focal Length Lens camera (Edmund Optics, 33301), respectively. We used custom programs to record and analyze behavior assays (open-field assay, elevated plus maze, Y-maze, and 3-chamber assay) using LabVIEW 2019 (National Instruments, Austin, TX, USA, v.190fl).

2.3. Grip Strength

Grip strength was recorded using an ALMEMO Universal Measuring Instrument (Holzkirchen, Germany, 24910). Grip strength was assessed during weeks 1 and 7 of Cr(VI) exposure. Rat forefeet were gently lowered to grip a horizontal bar 1.016 mm (diameter) by 6.35 cm (width). Rats were held horizontally by the tail and pulled backward at a steady rate until the rat released the grip of the horizontal bar [45]. Three trials were performed per rat, and the peak force exerted during each trial was recorded. Measurements were only recorded when the rats released grip from both forefeet simultaneously. Rats refusing to grip the bar after 10 attempts were recorded as noncompliant, and measurement was not recorded. Grip strength for each rat was measured in triplicate, and the median values were used for analyses and comparisons across study groups. Following each test, the instruments were cleaned and deodorized.

2.4. Open-Field Assay

The open-field assay was performed during weeks 2 and 8 of Cr(VI) exposure. The open-field chamber consisted of a square open area surrounded by four walls, with one transparent wall to enable side-view camera recording. The dimensions of the open-field chamber were 71 cm × 71 cm × 43 cm (L × W × H). Rats were individually placed in the center of the open-field chamber and allowed to freely explore the chamber for 10 min while being recorded from aerial and side views. Behavior recordings were assessed for changes in center area exploration (anxiety), distance traveled (locomotor function), and rearing behavior (motor coordination) [46]. The open-field chamber was cleaned with 70% ethanol between the recording sessions.

Center area exploration and distance traveled were measured using a program created in LabVIEW 2019. Rearing counts were scored manually from video recordings and defined as a rat standing on its hind legs and raising its forefeet above its center of mass; a rear was completed when the rat returned both forefeet to the floor of the chamber. Recorded trials were coded prior to manual scoring.

2.5. Elevated Plus Maze

The elevated plus maze was performed during weeks 3 and 9 of Cr(VI) exposure. The elevated plus maze consisted of four arms connected in the shape of a ‘plus’. Two arms were open (without walls), and two arms were enclosed on three sides by walls. Each arm was 51 cm × 15 cm (L × W), the closed arms had 43 cm high walls, and the middle of the maze contained a 15 cm × 15 cm (L × W) square. The entire maze was on a stand elevated 74.5 cm off the floor. Rats were individually placed in the middle of the maze, facing an open arm, and allowed to freely explore for 5 min while being recorded from an aerial view. Anxiety was assessed as the percentage of time spent exploring the open arm [47]. The maze was cleaned with 70% ethanol between the recording sessions. Decreased open-arm exploration indicates increased anxiety. Digital analyses of the time spent exploring the open arms of the elevated plus maze were performed using LabVIEW 2019.

2.6. Y-Maze

The Y-maze was performed during weeks 4 and 10 of Cr(VI) exposure. The Y-maze consisted of three radial arms angled in the shape of a ‘Y’, fully enclosed by walls. Each arm was 51 cm × 15 cm × 43 cm (L × W × H); arms were connected by an equilateral triangle with 15 cm sides. Rats were individually placed in the middle of the Y-maze and allowed to freely explore for 8 min while being recorded from an aerial view. Spatial memory was assessed as changes in non-alternating arm entry, while general activity was assessed as the frequency of arm entries [48]. The maze was cleaned with 70% ethanol between each recording session. Behavior was scored manually by tracking the sequence of arm entries to determine the number of alternations or non-alternations. An alternation was counted as all three arms entered in a sequence, whereas a non-alternation was counted as an arm entered more than once in a sequence of three entries. An entry was counted when all four paws crossed from the center triangle into an arm. Rats inherently explore novel areas; thus, increased non-alternations capitulate impaired spatial memory. The percentage of non-alternations was calculated using the following equation:

$$(\#NonAlternations/(\#Alternations+\#NonAlternations\left)\right)\times 100$$

Arm entries per minute were scored manually to assess activity. An arm entry was counted when all limbs left the center triangle and entered the arm. Increased arm entries per minute were interpreted as hyperactivity, while decreased arm entry frequency was interpreted as hypoactivity. Recorded trials were blinded prior to manual scoring.

2.7. 3-Chamber Assay

The 3-chamber assay was performed during weeks 5 and 11 of Cr(VI) exposure. The principle of this test is the free choice of test animals to spend time in any of the three chambers and interact with a familiar animal, empty cage, or unfamiliar animal placed under a basket [49]. The assay consisted of a large, enclosed space of 122 cm × 82 cm × 43 cm (L × W × H) and two interior walls with dividers to create three chambers, each 41 cm × 82 cm. Each of the exterior chamber contained a weighted inverted basket to hold a reference rat (cagemate or stranger), while the center chamber remained empty. Test rats were placed in the center chamber, and the dividers placed along the interior walls were removed before recording. Test rats were then allowed to freely explore the full chamber space and interact with the reference rats under the baskets. Test rats were recorded across three trials per test (10 min per trial) while being recorded from aerial and side cameras. Trial 1 was an acclimation phase with no additional rats; during trial 2, the cage mate was placed under one basket; during trial 3, the stranger rat was placed under the other basket. Recordings were analyzed manually, counting the number of entries into each chamber and the number of direct or indirect interactions. Direct interactions were counted as the test rat touching or climbing the baskets, while indirect interactions were counted as the test rat extending its body to within 5 cm of the basket. Recorded trials were blinded prior to manual scoring.

2.8. Statistical Analyses

The Anderson-Darling test was used to assess normality (α = 0.05). The Iterative Grubb’s Test and ROUT test were used to identify outliers (α = 0.05), which were removed where both tests agreed. Based on the normality test results, either a two-tailed, unpaired t-test with Welch’s correction or the Mann–Whitney test was used to assess statistical differences across Cr(VI) exposures and age groups. The criterion for statistical significance was p < 0.1 for all behavior assays. All analyses were conducted using GraphPad Prism 9 (v.9.5.1). Data in figures are expressed as mean ± standard error of the mean (SEM).

3. Results

3.1. Cr(VI) Did Not Affect Rat Body Mass

We recorded female rat body mass weekly during exposure. Cr(VI) exposure did not affect body mass, regardless of age or Cr(VI) concentration (Supplementary Figure S1).

3.2. Cr(VI) Altered Grip Strength in Older Age Groups

We assessed the effects of Cr(VI) on grip strength after 1 and 7 weeks of drinking water exposure (Figure 2). One geriatric control rat was identified as a statistical outlier (698 g) and was removed from comparisons at 7 weeks. When only considering Cr(VI) effects, not accounting for age groups, Cr(VI) had no effect on grip strength after either 1 or 7 weeks of exposure (Figure 2A,B). We then considered the effects of Cr(VI) across multiple age groups (Figure 2C,D). We observed no change in grip strength of young (3 months old), middle-aged (7 months old), or geriatric rats (18 months old) after 1 week of Cr(VI) exposure, and no differences due to age. After 7 weeks, we observed slightly decreased grip strength in young rats exposed to both Cr(VI) concentrations and a decrease in middle-aged rats exposed to 0.1 mg Cr(VI)/L: 415.2, 377.3, and 315.4 g for middle-aged control, 0.05, and 0.1 mg Cr(VI)/L, respectively. We observed significantly increased grip strength in geriatric rats after 7 weeks of exposure to 0.05 mg Cr(VI)/L (p = 0.01): 271.5, 359.5, and 323.1 g for control, 0.05, and 0.1 mg Cr(VI)/L, respectively (Figure 2D).

3.3. Cr(VI) Altered Locomotor Coordination in Middle-Aged Rats, Exploratory Behavior in Young Rats during the Open-Field Assay

Rats performed the open-field assay during weeks 2 and 8 of Cr(VI) exposure. We assessed exploratory behavior as the percentage of time spent exploring the center area of the open-field chamber (Figure 3), locomotor coordination as rearing behavior (Figure 4), and locomotor function as distance traveled (Figure 5).

We assessed center area exploration after 2 and 8 weeks of Cr(VI) exposure. One young control rat was identified as a statistical outlier (32.26%) and was removed from comparisons at 8 weeks. When considering the effects of Cr(VI) alone on exploratory behavior, we observed no change in center area exploration (Figure 3A,B). We then considered center area exploration across age groups (Figure 3C,D). After 2 weeks, we observed significantly increased center area exploration in young rats exposed to 0.1 mg Cr(VI)/L (p = 0.05) and slightly increased exploration in middle-aged rats exposed to 0.05 mg Cr(VI)/L, but we observed a slight decrease in geriatric rats exposed to Cr(VI) (Figure 3C). After 8 weeks, we observed increased center area exploration in young rats exposed to 0.05 mg Cr(VI)/L, whereas middle-aged rats exhibited a slight concentration-associated decrease and geriatric rats exhibited no effect (Figure 3D).

We assessed the effects of Cr(VI) on rearing behavior with no effect after 2 weeks of exposure but a slight decrease after 8 weeks of exposure to 0.05 mg Cr(VI)/L (Figure 4A,B). We then considered rearing behavior across age groups (Figure 4C,D). Rearing was not affected in young rats in either week. Middle-aged rats exhibited increased rearing after 2 weeks of Cr(VI) exposure, but this effect was reversed after 8 weeks with a statistically significant decrease after exposure to 0.05 mg Cr(VI)/L (p = 0.006). Geriatric rats exhibited slightly increased rearing after 2 weeks of exposure to 0.1 mg Cr(VI)/L, but no effect was apparent after 8 weeks.

We assessed the effects of Cr(VI) alone on distance traveled after 2 and 8 weeks of Cr(VI) exposure (Figure 5A,B). After 2 weeks, two young control rats were identified as statistical outliers (9509.78 and 12,880.69 cm) and removed from comparisons. After 8 weeks, two geriatric rats were identified as outliers (control = 18,404.30 cm; 0.1 mg/L = 10,453.75 cm) and were removed from comparisons. Considering these data regardless of age, Cr(VI) had no effect on distance traveled after 2 weeks, but we observed a significant decrease after 8 weeks of exposure to 0.05 mg Cr(VI)/L (p = 0.04): 5557 cm, 4436 cm, and 5242 cm for control, 0.05, and 0.1 mg Cr(VI)/L, respectively. We then considered the distance traveled across age groups (Figure 5C,D). After 2 weeks of Cr(VI) exposure, we observed a slight decrease in the distance traveled by young and geriatric rats but no effect in middle-aged rats (Figure 5C). After 8 weeks, the effect in young rats was not apparent, but we observed a decrease in geriatric rats exposed to 0.05 mg Cr(VI)/L (Figure 5D).

3.4. Cr(VI) Weakly Affected Anxiety in the Elevated Plus Maze

We assessed open-arm exploration in the elevated plus maze to measure anxiety-like behavior after 3 and 9 weeks of Cr(VI) exposure. When considering the effects of Cr(VI) alone, rats exposed to Cr(VI) for 3 weeks exhibited no change in open-arm exploration (Figure 6A). We observed a slight concentration-associated decrease in open-arm exploration after 9 weeks of Cr(VI) exposure: 25.6%, 21.5%, and 20.3% for control, 0.05, and 0.1 mg Cr(VI)/L, respectively (Figure 6B). We then assessed Cr(VI) effects across age groups (Figure 6C,D). We observed no change in open-arm exploration in rats of any age group after 3 weeks (Figure 6C). After 9 weeks, middle-aged rats still exhibited no effect, but young and geriatric rats exhibited a slight concentration-associated decrease in open-arm exploration: 41.5%, 39.0%, and 37.1% for young control, 0.05, and 0.1 mg Cr(VI)/L, respectively; 17.7%, 14.9%, and 9.7% for geriatric control, 0.05, and 0.1 mg Cr(VI)/L, respectively (Figure 6D).

3.5. Cr(VI) Affected Spatial Memory Distinctly across Ages, but No Effect on Activity during the Y-Maze

Rats performed the Y-maze after 4 and 10 weeks of Cr(VI) exposure. We assessed non-alternating exploration as a measure of spatial memory (Figure 7) and the frequency of arm entries as a measure of activity (Figure 8).

When considering the effects of Cr(VI) alone on spatial memory, we observed no change in non-alternations after 4 or 10 weeks of exposure (Figure 7A,B). We then assessed the effects of Cr(VI) on spatial memory across age groups (Figure 7C,D). We observed no Cr(VI) effects on non-alternations performed by young or middle-aged rats after 4 weeks of exposure, while geriatric rats exhibited a slight increase with Cr(VI) exposure. After 10 weeks, we observed a concentration-associated decrease in middle-aged females with a statistically significant decrease in the 0.1 mg Cr(VI)/L group (p = 0.02), but no effect in other ages: 43.4%, 36.3%, and 30.3% for control, 0.05, and 0.1 mg Cr(VI)/L groups, respectively (Figure 7D).

We assessed the number of arm entries per minute in the Y-maze as a measure of activity after 4 or 10 weeks of Cr(VI) exposure. We observed no effect after either week when we considered Cr(VI) exposure, regardless of age (Figure 8A,B). We then considered Cr(VI) effects on activity across age groups (Figure 8C,D). After 4 weeks, young and geriatric rats exhibited no Cr(VI) effect, whereas middle-aged rats became slightly hypoactive with Cr(VI) exposure (Figure 8C). After 10 weeks, middle-aged rats exhibited no change. Geriatric rats exhibited a slight decrease in activity following 0.05 mg Cr(VI)/L. Young rats exhibited a slight concentration-associated increase in arm entries per minute: 4.0, 4.1, and 4.3 arm entries per minute for control, 0.05, and 0.1 mg Cr(VI)/L, respectively (Figure 8D).

3.6. Cr(VI) Weakly Affected Sociability and Social Novelty Preference in the 3-Chamber Assay

We assessed sociability (Figure 9) and social novelty preference (Figure 10) in female rats after 5 and 11 weeks of Cr(VI) exposure using a 3-chamber assay.

To assess sociability, we considered the (1) number of interactions between a test rat and its cagemate, (2) number of interactions between a test rat and an empty cage, and (3) the ratio of cagemate to empty cage interactions (Figure 9, Supplementary Figure S2). We first considered the effects of Cr(VI) on sociability regardless of age (Figure 9A,B; Supplementary Figure S2A,B). We observed that female rats overall preferred interacting with a cagemate instead of an empty cage. After 5 weeks, we observed no change in cagemate or empty cage interactions. After 11 weeks, we still observed no change in cagemate interactions, but Cr(VI)-exposed rats exhibited a slight concentration-associated increase in empty cage interactions: 5.4, 6.1, and 7.1 empty cage interactions for control, 0.05, and 0.1 mg Cr(VI)/L groups, respectively (Figure 9B). As a result of the increased number of empty cage interactions in the 0.1 mg Cr(VI)/L group, there appeared to be a loss in preference between interacting with a cagemate vs. an empty cage. However, when we assessed these data as a ratio per individual rat, we instead observed an increase in sociability ratio (cagemate/empty cage) for the 0.05 mg Cr(VI)/L group and no effect in the 0.1 mg Cr(VI)/L group (Supplementary Figure S2B).

We then considered the effects of Cr(VI) on the sociability of rats across age groups (Figure 9C,D; Supplementary Figure S2C,D). After 5 weeks, we observed a general preference for interacting with the cagemate, except for middle-aged rats exposed to 0.1 mg Cr(VI)/L; this group exhibited fewer interactions with cagemates relative to control or 0.05 mg Cr(VI)/L groups, and the SEM overlapped between interactions with cagemates and the empty cage (Figure 9C). We further observed increased cagemate and empty cage interactions by Cr(VI) exposed geriatric rats: 7.6, 10.6, and 9.9 cagemate interactions and 4.0, 6.7, and 5.2 empty cage interactions for control, 0.05, and 0.1 mg Cr(VI), respectively. We observed no effect in young rats after 5 weeks of Cr(VI) exposure. When we considered these data as a ratio for each rat, we observed that the middle-aged rats shifted closer to a ratio of 1.00 with Cr(VI) exposure, indicating reduced sociability; mean ratios were 1.92, 1.84, and 1.26 for control, 0.05, and 0.1 mg Cr(VI)/L, respectively (Supplementary Figure S2C).

For 11 weeks sociability by age, we observed a similar overall preference for interacting with cagemates, except for young or middle-aged rats exposed to 0.1 mg Cr(VI)/L, which exhibited no preference between the cagemate and empty cage (Figure 9D). In young rats, we still observed no effect in cagemate interactions, whereas empty cage interactions increased: 8.4, 9.0, and 12.0 empty cage interactions for control, 0.05, and 0.1 mg Cr(VI)/L groups, respectively (Figure 9D). In middle-aged rats exposed to 0.1 mg/L we observed a slight decrease in cagemate interactions. Geriatric rats exhibited slightly more interactions with both cagemate and empty cages after Cr(VI) exposure, with the increase in empty cage interactions more noticeable: 5.2, 6.6, 6.6 cagemate interactions and 1.9, 3.5, and 4.2 empty cage interactions for control, 0.05, and 0.1 mg Cr(VI)/L, respectively (Figure 9D). When we considered these data as a ratio by individual rats, we observed that the ratios for young and geriatric rats increased after 0.05 mg Cr(VI)/L (Supplementary Figure S2D).

Altogether, these results suggest that Cr(VI) reduced sociability in middle-aged rats after 5 or 11 weeks and in young and geriatric rats after 11 weeks.

To assess social novelty preference, we examined (1) the number of interactions between a test rat and its cagemate, (2) the number of interactions between a test rat and a stranger rat, and (3) the ratio of cagemate to stranger-rat interactions during the 3-chamber assay (Figure 10, Supplementary Figure S3). We first considered the effects of Cr(VI) on social preference, regardless of age (Figure 10A,B). Across all groups, we observed a strong preference for interacting with a stranger rat after 5 and 11 weeks, indicating a strong preference for social novelty. After 5 weeks, we observed no changes in interactions with a cagemate or stranger rat. After 11 weeks, we observed a slight increase in stranger interactions: 9.6, 10.3, and 11.3 stranger interactions for control, 0.05, and 0.1 mg Cr(VI)/L, respectively. When considering these data as a ratio (cagemate/stranger), we observed no change after 5 weeks and a statistically significant decrease in the 0.05 mg Cr(VI)/L group after 11 weeks (Supplementary Figure S3A,B).

We then considered Cr(VI) effects on social preference across age groups (Figure 10C,D; Supplementary Figure S3C,D). After 5 weeks, we observed that young rats exhibited a concentration-associated increase in stranger-rat interactions with no change in cagemate interactions: 11.8, 14.3, and 15.2 stranger interactions for control, 0.05, and 0.1 mg Cr(VI)/L, respectively. We observed no change in cagemate interactions by middle-aged rats after 5 weeks but a slight decrease in stranger interactions in the 0.05 mg Cr(VI)/L group. We observed a slight concentration-associated decrease in cagemate interactions by geriatric rats and a slight decrease in stranger-rat interactions by geriatric rats exposed to 0.1 mg Cr(VI)/L (Figure 10C). When we considered these data as a ratio, we observed no Cr(VI) effect in young rats, an increased ratio in middle-aged rats exposed to 0.05 mg Cr(VI)/L, and a decreased ratio in geriatric rats exposed to both Cr(VI) concentrations (Supplementary Figure S3C,D).

After 11 weeks, we again observed young rats exhibited a concentration-associated increase in stranger-rat interactions with no change in cagemate interactions: 12.8, 14.5, and 15.5 stranger interactions for control, 0.05, and 0.1 mg Cr(VI)/L (Figure 10D). We observed no differences in cagemate or stranger interactions in middle-aged rats after 11 weeks of Cr(VI) exposure. In geriatric rats, we observed decreased cagemate interactions after 0.05 mg Cr(VI)/L and increased stranger interactions after 0.1 mg Cr(VI)/L (Figure 10D). When we considered these data as a ratio, we observed little to no effect in young or middle-aged rats, whereas geriatric rats exhibited a decreased ratio after exposure to 0.05 mg Cr(VI)/L.

Altogether, these results suggest Cr(VI) exposure increased social novelty preference in young rats after 5 and 11 weeks, but decreased this behavior in geriatric rats.

3.7. Cr(VI) Behavior Effects Are Distinct across Sexes

We previously reported results from male rats in this study [44]. Here, we report behaviors where we observed notable sex differences after Cr(VI) exposure (Figure 11). After 2 weeks, we observed that middle-aged female rats exposed to 0.05 mg Cr(VI)/L exhibited increased center exploration, whereas males exposed to the same concentration exhibited significantly decreased center exploration (Figure 11A). After 2 weeks, young female rats exhibited little to no Cr(VI) impact on distance traveled, whereas male rats exhibited significantly decreased distance traveled in the 0.05 mg Cr(VI)/L group (Figure 11B). After 7 weeks, we observed a concentration-associated decrease in female grip strength with statistical significance in the 0.1 mg Cr(VI)/L group, whereas males exhibited significantly increased grip strength in the 0.05 mg Cr(VI)/L group (Figure 11C). After 8 weeks, we observed a statistically significant decrease in female rearing but no effect in males (Figure 11D). After 9 weeks, we observed a slight decrease in female open-arm exploration following 0.05 mg Cr(VI)/L during the elevated plus maze, whereas males exhibited a statistically significant decrease in the 0.1 mg Cr(VI)/L group (Figure 11E). After 10 weeks, we observed a concentration-associated decrease in female alternations in the Y-maze, whereas males exhibited a concentration-associated increase; both groups were statistically significant in the 0.1 mg Cr(VI)/L groups (Figure 11F). After 10 weeks, we also observed activity in the Y-maze was unaffected in middle-aged females, whereas males exhibited significantly decreased activity with Cr(VI) exposure (Figure 11G). After 11 weeks, middle-aged females exhibited fewer cagemate interactions in the 0.1 mg Cr(VI)/L group with no impact on empty cage interactions, whereas males exhibited significantly decreased interactions with both in the 0.05 mg Cr(VI)/L group and slightly decreased interactions with both in the 0.1 mg Cr(VI)/L group (Figure 11H). Also, after 11 weeks, we observed a stronger preference for social novelty in female rats, regardless of age, than in male rats (Figure 11I). After Cr(VI) exposure, females exhibited a slight concentration-associated increase in stranger-rat interactions and no impact on cagemate interactions, whereas males exposed to 0.05 mg Cr(VI)/L exhibited the same number of interactions with both cagemates and strangers. We further observed a sex difference in this assay for the young rats (Figure 11J). Young female rats exhibited a slight concentration-associated increase in stranger-rat interactions and no impact on cagemate interactions, whereas young male rats exhibited more interactions with cagemates after exposure to 0.05 mg Cr(VI)/L with no impact on stranger interactions and increased stranger interactions in the 0.1 mg Cr(VI)/L group with no impact on cagemate interactions.

4. Discussion

This study describes behavior effects in female Sprague-Dawley rats across three different ages (3, 7, and 18 months old) after a 90-day exposure to Cr(VI) in drinking water at WHO and US EPA maximum contaminant levels (0.05 and 0.1 mg/L, respectively). Critically, we used Cr(VI) drinking water levels that are lower than any other Cr(VI) neurotoxicity study reported thus far, and these levels are drastically lower than the drinking water levels reported to induce cancer in mice and rats (180 mg/L) [50]. We report age differences in behavior effects and, in some cases, opposite effects, highlighting the need to consider multiple life stages in toxicology studies. We previously reported behavior effects in male rats using the same model and exposure parameters, and here, we presented behavior data where we saw stark sex differences [44].

A limited number of publications have considered Cr(VI) behavioral effects in rodents [39,41,42,51,52,53]. Most assessed locomotor activity (rotarod test, grid floor activity cage, and open-field assay), providing a consensus that Cr(VI) induces locomotor deficits in rodents. Estrela et al. [41] provided evidence of social memory impairments due to Cr(VI) in tannery effluent but reported no impacts on olfactory function. Hegazy et al. [42] assessed Cr(VI) impacts in the novel object recognition after intranasal exposure to 0.125, 0.25, 0.5 mg Cr(VI)/kg/d; they reported impaired learning/memory in the 0.5 mg group as early as 2 weeks, and significantly impaired learning/memory at all concentrations tested after 1 or 2 months of exposure. Sedik et al. [53] also performed an open-field assay and reported a significant reduction in rearing counts per minute following 2 mg/kg potassium dichromate administered intranasally.

4.1. Behavior Effects of Cr(VI), Independent of Age

Our results indicate behavior effects at very low concentrations of Cr(VI) in drinking water, levels that are considered safe for widespread and chronic human consumption. These data are summarized in Supplementary Table S1. Considering the effects of Cr(VI) alone, we first observed behavioral changes in females after 8 weeks of exposure with significantly decreased distance traveled in the 0.05 mg Cr(VI)/L group. We also observed a slight decrease in rearing activity following 8 weeks of Cr(VI) exposure. Together, these data suggest Cr(VI) impairs locomotor function. After 9 weeks of Cr(VI) exposure, we observed a slight concentration-associated decrease in open-arm exploration in the elevated plus maze, suggesting Cr(VI) contributes to anxiogenic effects. After 11 weeks, we observed two changes in the 3-chamber assay. During the sociability trial, we observed a concentration-associated increase in empty cage interactions, which resulted in less discrimination between cagemates and empty cages, suggesting Cr(VI) decreased sociability. During the social preference trial, we observed slightly increased interactions with the stranger rat, suggesting Cr(VI) increased preference for social novelty.

Here, we discuss sex differences in Cr(VI) behavior effects by comparing the effects we observed in females with effects we previously reported in males [44]. These data are summarized in Supplementary Table S2. While we first observed significant behavior effects in females after 8 weeks of exposure, we observed effects in males as early as week 2 of Cr(VI) exposure. In addition to differences in the temporal manifestation of Cr(VI) neurotoxicity, the behaviors impacted and the degree of change in behavior were distinct between the sexes. After 2 weeks of exposure in males, we observed decreased rearing behavior and distance traveled, though we did not observe this effect in females until 8 weeks of exposure. After 8 weeks, we observed decreased rearing and distance traveled in both sexes, but this effect was only statistically significant in 0.05 mg Cr(VI)/L exposed females. We observed a statistically significant impairment of spatial memory in males after 4 weeks, while female spatial memory was unaffected after 4 or 10 weeks. After 10 weeks, males became hypoactive, whereas female activity was unaffected. We observed slightly decreased sociability in males after 5 weeks, which we did not observe in females until 11 weeks. We observed that 11 weeks of exposure to 0.05 mg Cr(VI)/L in males resulted in no social novelty preference, but females exhibited a stronger preference for social novelty. Overall, we observed that locomotor effects were more prominent in Cr(VI)-exposed females, but Cr(VI)-exposed males exhibited greater impacts on memory, sociability, and social novelty preference. Though behavior effects manifested distinctly between males and females, we observed behavior changes in males 6 weeks earlier than in females, suggesting males may be more vulnerable to Cr(VI) neurotoxicity.

4.2. Heads: Impact of Age on Cr(VI) Neurotoxicity

Ours is the first study to consider different ages in Cr(VI) neurotoxicity, and these data are summarized in Supplementary Table S3. Throughout this study, we observed notable age differences in Cr(VI) effects on female grip strength, locomotor coordination, anxiety, spatial memory, sociability, and social novelty preference. In the context of our previous study, we observed sex differences in Cr(VI) effects on these behaviors in rats of the same age (e.g., young females vs. young males). Here, we discuss age and sex differences in Cr(VI) neurotoxicity.

Cr(VI) had no effect on female grip strength after 1 week, but we observed age differences in Cr(VI) effects on grip strength after 7 weeks. Young and middle-aged females exhibited decreased grip strength, directly contrasting a significant increase in geriatric females. Regarding sex differences, males exhibited significant changes in grip strength after 1 and 7 weeks of Cr(VI) exposure. Young males also exhibited decreased grip strength after 1 week of exposure, and this decrease was significant after 7 weeks, but exhibited a stronger effect than young females. After 7 weeks, middle-aged males exposed to 0.05 mg Cr(VI)/L exhibited significantly increased grip strength, which contrasted the decrease we observed for females exposed to 0.05 mg Cr(VI)/L. Geriatric males exhibited a concentration-associated increase in grip strength at both time points, which was a stronger effect than what we observed in females. Age-related changes in Cr(VI) effects on grip strength are intriguing and puzzling, though the effects are mostly consistent across sexes. Cr(VI) impaired grip strength in male and female young rats but increased grip strength in male and female geriatric rats. It is possible that an age-related decrease in stomach and ileum pH may enhance the reduction of Cr(VI) to Cr(III) [54]. Some studies have reported that Cr(III) supplementation may be beneficial to muscle performance, thus improving grip strength in geriatric rats [55].

We assessed locomotor function using an open-field assay and observed age differences in female rats exposed to Cr(VI) for motor coordination (rearing) and locomotor function (distance traveled). We observed no change in rearing by young females after 2 or 8 weeks, but we observed an increase in middle-aged and geriatric females after 2 weeks. After 8 weeks, this effect was not apparent in geriatric females and reversed in middle-aged females. We observed no change in distance traveled by females in any age group after 2 weeks, but we observed slightly decreased distance traveled in geriatric females exposed to 0.05 mg Cr(VI)/L after 8 weeks. Regarding sex differences in locomotor function, we observed overall greater rearing counts and distance traveled by females compared to males. Cr(VI) decreased rearing in young males after 2 and 8 weeks of exposure, contrasting the lack of an effect in young females. While sex differences in young rats’ rearing behavior are not reported in the Cr(VI) literature, other data suggest young females may be less vulnerable to chemical-induced locomotor impairment than young males [56,57]. In middle-aged rats, we observed a greater reduction in rearing behavior following Cr(VI) exposure in females than in males. These data may suggest vulnerability to the effects of Cr(VI) on locomotor function changes with age, and this age-related vulnerability is distinct between sexes.

We observed age-related differences in anxiety using the open-field assay and elevated plus maze. After 2 weeks of Cr(VI) exposure, we observed increased center area exploration in the open-field assay by young females exposed to 0.1 mg/L and middle-aged females exposed to 0.05 mg/L, but a slight decrease in geriatric females exposed to 0.1 mg/L. During the later open-field test (8 weeks), we observed an attenuation in the Cr(VI) effect on young females, no apparent effect in geriatric females, and a reversed effect in middle-aged females exhibiting less center exploration. These data suggest shorter Cr(VI) exposure can induce anxiolytic effects in young and middle-aged females, but anxiogenic effects in geriatric females, whereas longer Cr(VI) exposures may induce an anxiogenic effect in middle-aged females. Our previous report is the only Cr(VI) neurotoxicity study that includes Cr(VI) effects on anxiety, and we observed some sex differences. Similar to females, young male rats also exhibited an anxiogenic effect after 2 weeks of exposure to Cr(VI), however, this was apparent in the 0.05 mg/L male group vs. the 0.1 mg/L group in females; middle-aged males exhibited the opposite effect as females, with an anxiogenic effect apparent in the 0.05 mg/L males vs. anxiolytic in females. We further assessed anxiety using the elevated plus maze, where Cr(VI) exhibited anxiogenic effects in geriatric females after 9 weeks but no effect in young or middle-aged females. In contrast, we reported anxiogenic effects in young and middle-aged male rats after 9 weeks. In sum, these data suggest Cr(VI) exhibits variable effects on anxiety that are age-, sex-, time-, and context-specific.

Our spatial memory assessment in the Y-maze is potentially the most interesting result of this study. We observed no Cr(VI) effects in females after 4 or 10 weeks of exposure, but males exhibited a concentration-associated increase in non-alternations after 4 weeks. Considering age groups at week 4, we observed no effects across all female age groups, while the male Cr(VI) effect was primarily driven by the young and geriatric male groups. After 10 weeks Cr(VI) exposure, we observed middle-aged females exhibited a concentration-associated decrease in non-alternations after 10 weeks Cr(VI) exposure, which directly contrasted with a concentration-associated increase in middle-aged males. These data suggest Cr(VI) exposure improved spatial memory in middle-aged females but impaired spatial memory in middle-aged males. Instead, we posit middle-aged females developed a turning side preference from Cr(VI)-induced hippocampal damage, a phenomenon where severe hippocampal damage results in decreased spontaneous non-alternations in the Y-maze [58,59].

We observed some age- and sex-differences in rat activity, as measured by the number of arm entries per minute during the Y-maze. Females were generally more active than males when comparing across age groups. After 4 weeks of Cr(VI) exposure, middle-aged females became slightly hypoactive with no apparent effects in young or geriatric females, whereas young and geriatric males were slightly hyperactive after Cr(VI) exposure, and middle-aged males were slightly hypoactive. After 10 weeks of Cr(VI) exposure, young females became hyperactive, and there was no apparent effect in older females. In males, we observed middle-aged and geriatric rats became hypoactive after 10 weeks, whereas young rats were still slightly hyperactive. Other studies reported Cr(VI) exposure decreased motor activity in the motor activity cage assay [42,51,52,53]. Hyperactivity in rodents is an autism spectrum disorder phenotype, which is linked to Cr(VI) exposure in human populations [33,60]. Evidence also suggests metal exposure induces hyperactivity in rats, which is likely due to neuropathology in the cerebellum and striatum [61]. Cr(VI) induced neuropathology in the rat, guinea pig, and chicken cerebellum, suggesting cerebellar damage may induce hyperactivity after Cr(VI) exposure [40,62,63]. Cr(VI)-induced neuropathology in the striatum has not been reported.

Cr(VI) altered sociability and social novelty preference distinctly across age groups in the 3-chamber assay. Cr(VI) reduced sociability in middle-aged rats (both sexes) after 5 weeks; after 11 weeks, we observed decreased sociability in all Cr(VI)-exposed female rats, while effects in males were more variable. During the social novelty preference trial, we observed females generally had a stronger preference for social novelty than males. In the Cr(VI)-exposed groups, we observed increased social novelty preference in young females after 5 and 11 weeks and in geriatric females after 11 weeks. In males, we observed a reduced preference for social novelty with middle-aged and geriatric males exposed to 0.05 mg Cr(VI)/L for 11 weeks. The 3-chamber assay is used to assess autism spectrum disorder and developmental disorder phenotypes in rodent models, typically demonstrated by reduced sociability [64]. However, an increased preference for social novelty following a neurotoxic exposure (observed in geriatric rats of both sexes) is not widely documented. One group described inhibiting the basolateral amygdala impaired communication with the ventral hippocampus and increased social interactions, while another suggested antagonism of the dorsal raphe increased sociability, identifying three brain regions of interest for future studies in geriatric rats [65,66]. One other report discussed changes in social memory following exposure to Cr(VI)-laden tannery effluent in 2–3 month-old female Swiss mice [41]. This group reported exposure to tannery effluent increased anogenital exploration and chasing times of novel mice, suggesting impairment of social memory in exposed mice. Estrela et al. [41] also reported that co-treatment with vitamin C alleviated these effects. Vitamin C is the key reducing agent for Cr(VI), thus suggesting Cr(VI) in tannery effluent is the primary neurotoxicant [67]. Estrela et al. [41] reported changes in social memory, while our study assessed changes in sociability and social preference. While we did not assess social memory in this manner, preference for social novelty may be influenced by the ability to store and recall social memory [68].

4.3. Tails: Impact of Cr(VI) Neurotoxicity on Aging

Growing evidence suggests Cr(VI) induces aging phenotypes in cell culture models and human populations, though no studies have assessed how Cr(VI) affects brain aging [27,28,29,30,31]. Here, we consider evidence for aging behavior phenotypes in our results for Cr(VI) behavior effects by focusing on behavior differences across control rats of each age group (3, 7, or 18 months old) and looking for similar effects across the Cr(VI) groups. We previously reported Cr(VI) acted as a gerontogen in male rats and induced premature aging behavior phenotypes in the grip strength assay, elevated plus maze, and Y-maze. Here, we report Cr(VI)-altered behavior in an aging-related manner in an open-field assay and Y-maze in female rats.

Reports show center area exploration, rearing behavior, and distance traveled in the open-field assay decrease with age in healthy male mice (7, 46, and 72 weeks old) [69]. In the open-field assay, we observed an age-associated decrease in rearing behavior after 2 and 8 weeks and an age-associated decrease in center area exploration after 8 weeks. After 8 weeks, Cr(VI) reduced both behaviors in middle-aged females, suggesting Cr(VI) may induce an aging behavioral phenotype in middle-aged females.

Multiple studies have considered arm entries per minute in the Y-maze as a measure of activity, and at least one study demonstrated exposing mice to a gerontogen (d-galactose) reduced mouse activity [70]. We observed an age-associated decrease in activity during the Y-maze after 4 and 10 weeks. We observed a weak hypoactive effect in middle-aged rats exposed to both concentrations of Cr(VI) after 4 weeks of exposure but no effect after 10 weeks. Middle-aged males also became significantly hypoactive after 4 and 10 weeks of exposure to 0.05 mg Cr(VI)/L. These data suggest Cr(VI) may induce an age-related change in activity in male and female rats.

5. Conclusions

This report described behavior effects in female rats after exposure to environmentally relevant concentrations of Cr(VI) in drinking water—concentrations that are considered safe for chronic, widespread human consumption. Further, we described how behavior effects of Cr(VI) manifest distinctly across age groups (young, middle-aged, geriatric) and compared these data to our previous report in males to highlight sex differences. Results from this study emphasize the importance of considering age and sex differences in Cr(VI) neurotoxicity, as well as in broader toxicology contexts. Given that rodents endogenously synthesize vitamin C (a key reducer of Cr[VI]) and the adjustment factors for risk assessment from rodent studies to humans, our results indicate a need to revisit and/or revise the current regulations for Cr(VI) in drinking water.