University of Louisville
Using a Toxic Aging Coin Approach to Investigate How Low Concentrations of Hexavalent Chromium in Drinking Water Induce Blood-Brain Barrier Damage
Grade Level at Time of Presentation
Junior
Major
Neuroscience
Institution 24-25
University of Louisville
KY House District #
32
KY Senate District #
6
Faculty Advisor/ Mentor
John P. Wise, Jr.
Department
Pediatrics Research Institute, Department of Pediatrics, University of Louisville
Abstract
Introduction:
We are currently facing an aging crisis, as 20% of the U.S. population will be geriatric (65+) by 2030. This growing geriatric population will result in an increasing prevalence of age-related diseases, coinciding with ubiquitous environmental pollution. Environmental pollution threatens the health of people at all ages and contributes to disease etiology. Geriatric individuals are often more susceptible to the negative health effects from environmental pollution, yet we have a poor understanding of age differences in toxicology. We use a Toxic Aging Coin approach to investigate: 1) how age impacts toxicity (heads), and 2) how chemicals accelerate aging (tails).. Cr(VI) is a widespread environmental pollutant used in a variety of industrial processes such as cement production, dye-making, and increasingly in solar panel manufacturing. Drinking water Cr(VI) contamination may originate from industrial effluents or from natural sources such as ultramafic bedrock. Cr(VI) is a known lung carcinogen via inhalation and its toxicity in peripheral tissues is well-known, but significant knowledge gaps persist for Cr(VI) neurotoxicity. In humans, Cr(VI) contributes to a variety of neurological effects, such as polyneuropathy, motor neuron disease, and autism spectrum disorder, though underlying mechanisms are unknown. Results from animal studies similarly lack extensive behavioral analyses and key mechanistic data, and further do not identify brain regions or cell types that are particularly vulnerable to Cr(VI). We recently reported rats exposed to Cr(VI) in drinking water exhibited multiple behavioral effects, including anxiety, grip strength, and spatial memory, with distinct age- and sex-differences. Preliminary data from our lab indicate Cr(VI) targets the hippocampus, with evidence for marked neurodegeneration and neuroinflammation. However, it is unclear if these are direct effects of Cr(VI) on these cells, or secondary effects from damage to the blood-brain barrier. The blood-brain barrier (BBB) is a critical structure to protect the brain parenchyma; it allows for the selective transport of key materials into the brain while preventing penetration of noxious materials (e.g. inflammatory cytokines, immune cells, toxicants, and pathogens). Age is a key factor, as the BBB is not completely formed until after birth and naturally becomes leakier with age. Further, chronic inflammation and damage from toxicants can accelerate BBB deterioration and contribute to neurological diseases. Key components of the BBB are tight junction proteins (e.g. occludin and claudin-5) between endothelial cells which regulate the passage of materials through the capillary epithelium. Tissue Plasminogen Activator (tPA) converts plasminogen to its active form, plasmin, a serum protease linked to inflammation. Additionally, decreased levels of tPA have been linked to both aging and amyloid beta accumulation. We hypothesize that Cr(VI) targets endothelial cells and pericytes, resulting in BBB damage and dysfunction.
Methods:
We exposed Sprague-Dawley rats (both sexes) from three ages (3-, 7-, and 18-months-old) to low concentrations of Cr(VI) in drinking water (0.05 and 0.1 mg/L) for 90 days. Importantly, 0.05 and 0.1 mg/L correspond to the WHO and U.S. EPA maximum contaminant levels for drinking water Cr, respectively. After 90 days we harvested the brains and microdissected one hemisphere for inductively coupled plasma-mass spectrometry (ICP-MS) analyses. The other hemisphere was preserved to assess BBB integrity and protein expression in the dorsal hippocampus by immunofluorescence (i.e., claudin-5, occludin, albumin, tPA, and glial fibrillary acidic protein [GFAP]). Changes in claudin-5 and occludin expression indicate structural abnormalities within tight junctions, while GFAP marks astrocytes and can indicate astrocyte activation. Presence of albumin in the brain parenchyma indicates BBB leakage, while tPA expression can indicate capillary inflammation. Three tissues per animal were assessed, both at the whole-hippocampus level as well as at 60x magnification to observe capillary structure. Protein expression was quantified via ROI analyses and data were reported as fold-change relative to control.
Results:
We assessed Cr accumulation across brain regions via ICP-MS; Cr accumulated only in the hippocampus, especially the hippocampus of geriatric and middle-aged female rats. We assessed essential metal levels (Fe, Cu, Se, Mg, Co, Mn, Zn) in the hippocampus via ICP-MS, observing opposite effects between sexes and opposite effects between young vs. older rats within the same sex. Young male rats exhibited mostly increased essential metals, with significantly increased Fe and Cu levels; while young females exhibited mostly decreased essential metals, with significantly decreased Fe and Mg levels. In contrast, essential metals were significantly decreased in the hippocampus of middle-aged and geriatric male rats, while these were significantly increased in the hippocampus of middle-aged and geriatric female rats. tPA expression was decreased in the hippocampus of geriatric female rats exposed to 0.1 mg/L Cr(VI). GFAP expression was increased in Cr(VI)-exposed geriatric female rats at the whole hippocampus level but decreased at the capillary level.
Conclusions:
ICP-MS analyses indicate Cr(VI) induced essential metal dyshomeostasis with clear sex differences and Cr selectively accumulated in the hippocampus of aged females, particularly geriatric females. Elevated GFAP expression suggests astrocyte activation in the hippocampus of geriatric female rats, whereas decreased expression around blood vessels may be caused by astrocyte end-feet retraction, which can impair the integrity of the BBB by removing structural support, potentially weakening the BBB. Decreased tPA expression may indicate an exacerbated aging effect which may indicate an Alzheimer’s-like phenotype in geriatric female rats due to its role in amyloid beta removal. Future directions will further examine and clarify Cr(VI)’s effects on the BBB, particularly at the capillary level, including replication of tPA and GFAP assessments across age groups. Support from R21ES033327 (JPWJr), T32ES011564 (STV), University of Louisville Summer Research Opportunity Program (WJB).
Using a Toxic Aging Coin Approach to Investigate How Low Concentrations of Hexavalent Chromium in Drinking Water Induce Blood-Brain Barrier Damage
Introduction:
We are currently facing an aging crisis, as 20% of the U.S. population will be geriatric (65+) by 2030. This growing geriatric population will result in an increasing prevalence of age-related diseases, coinciding with ubiquitous environmental pollution. Environmental pollution threatens the health of people at all ages and contributes to disease etiology. Geriatric individuals are often more susceptible to the negative health effects from environmental pollution, yet we have a poor understanding of age differences in toxicology. We use a Toxic Aging Coin approach to investigate: 1) how age impacts toxicity (heads), and 2) how chemicals accelerate aging (tails).. Cr(VI) is a widespread environmental pollutant used in a variety of industrial processes such as cement production, dye-making, and increasingly in solar panel manufacturing. Drinking water Cr(VI) contamination may originate from industrial effluents or from natural sources such as ultramafic bedrock. Cr(VI) is a known lung carcinogen via inhalation and its toxicity in peripheral tissues is well-known, but significant knowledge gaps persist for Cr(VI) neurotoxicity. In humans, Cr(VI) contributes to a variety of neurological effects, such as polyneuropathy, motor neuron disease, and autism spectrum disorder, though underlying mechanisms are unknown. Results from animal studies similarly lack extensive behavioral analyses and key mechanistic data, and further do not identify brain regions or cell types that are particularly vulnerable to Cr(VI). We recently reported rats exposed to Cr(VI) in drinking water exhibited multiple behavioral effects, including anxiety, grip strength, and spatial memory, with distinct age- and sex-differences. Preliminary data from our lab indicate Cr(VI) targets the hippocampus, with evidence for marked neurodegeneration and neuroinflammation. However, it is unclear if these are direct effects of Cr(VI) on these cells, or secondary effects from damage to the blood-brain barrier. The blood-brain barrier (BBB) is a critical structure to protect the brain parenchyma; it allows for the selective transport of key materials into the brain while preventing penetration of noxious materials (e.g. inflammatory cytokines, immune cells, toxicants, and pathogens). Age is a key factor, as the BBB is not completely formed until after birth and naturally becomes leakier with age. Further, chronic inflammation and damage from toxicants can accelerate BBB deterioration and contribute to neurological diseases. Key components of the BBB are tight junction proteins (e.g. occludin and claudin-5) between endothelial cells which regulate the passage of materials through the capillary epithelium. Tissue Plasminogen Activator (tPA) converts plasminogen to its active form, plasmin, a serum protease linked to inflammation. Additionally, decreased levels of tPA have been linked to both aging and amyloid beta accumulation. We hypothesize that Cr(VI) targets endothelial cells and pericytes, resulting in BBB damage and dysfunction.
Methods:
We exposed Sprague-Dawley rats (both sexes) from three ages (3-, 7-, and 18-months-old) to low concentrations of Cr(VI) in drinking water (0.05 and 0.1 mg/L) for 90 days. Importantly, 0.05 and 0.1 mg/L correspond to the WHO and U.S. EPA maximum contaminant levels for drinking water Cr, respectively. After 90 days we harvested the brains and microdissected one hemisphere for inductively coupled plasma-mass spectrometry (ICP-MS) analyses. The other hemisphere was preserved to assess BBB integrity and protein expression in the dorsal hippocampus by immunofluorescence (i.e., claudin-5, occludin, albumin, tPA, and glial fibrillary acidic protein [GFAP]). Changes in claudin-5 and occludin expression indicate structural abnormalities within tight junctions, while GFAP marks astrocytes and can indicate astrocyte activation. Presence of albumin in the brain parenchyma indicates BBB leakage, while tPA expression can indicate capillary inflammation. Three tissues per animal were assessed, both at the whole-hippocampus level as well as at 60x magnification to observe capillary structure. Protein expression was quantified via ROI analyses and data were reported as fold-change relative to control.
Results:
We assessed Cr accumulation across brain regions via ICP-MS; Cr accumulated only in the hippocampus, especially the hippocampus of geriatric and middle-aged female rats. We assessed essential metal levels (Fe, Cu, Se, Mg, Co, Mn, Zn) in the hippocampus via ICP-MS, observing opposite effects between sexes and opposite effects between young vs. older rats within the same sex. Young male rats exhibited mostly increased essential metals, with significantly increased Fe and Cu levels; while young females exhibited mostly decreased essential metals, with significantly decreased Fe and Mg levels. In contrast, essential metals were significantly decreased in the hippocampus of middle-aged and geriatric male rats, while these were significantly increased in the hippocampus of middle-aged and geriatric female rats. tPA expression was decreased in the hippocampus of geriatric female rats exposed to 0.1 mg/L Cr(VI). GFAP expression was increased in Cr(VI)-exposed geriatric female rats at the whole hippocampus level but decreased at the capillary level.
Conclusions:
ICP-MS analyses indicate Cr(VI) induced essential metal dyshomeostasis with clear sex differences and Cr selectively accumulated in the hippocampus of aged females, particularly geriatric females. Elevated GFAP expression suggests astrocyte activation in the hippocampus of geriatric female rats, whereas decreased expression around blood vessels may be caused by astrocyte end-feet retraction, which can impair the integrity of the BBB by removing structural support, potentially weakening the BBB. Decreased tPA expression may indicate an exacerbated aging effect which may indicate an Alzheimer’s-like phenotype in geriatric female rats due to its role in amyloid beta removal. Future directions will further examine and clarify Cr(VI)’s effects on the BBB, particularly at the capillary level, including replication of tPA and GFAP assessments across age groups. Support from R21ES033327 (JPWJr), T32ES011564 (STV), University of Louisville Summer Research Opportunity Program (WJB).