Nephrotoxic Potential of Putative 3,5-Dichloroaniline (3,5-DCA) Metabolites and Biotransformation of 3,5-DCA in Isolated Kidney Cells from Fischer 344 Rats
Abstract
:1. Introduction
2. Results
2.1. Time and Concentration Cytotoxicity Relationships for 3,5-DCA Metabolites
2.2. Renal Biotransformation and 3,5-DCNB Cytotoxicity
2.3. Antioxidants and 3,5-DCPHA Cytotoxicity
2.4. 3,5-DCA Metabolism by IKC
2.5. Effect of Diethyldithiocarbamic Acid (DEDTCA) on 3,5-DCA Metabolism
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Chemicals
4.3. Preparation and Treatment of Isolated Kidney Cells
4.4. IKC Metabolism of 3,5-DCA
4.4.1. IKC Incubations and Metabolite Isolation
4.4.2. Determination of Sulfate and Glucuronide Conjugates
4.4.3. Inhibition of CYP2C Metabolism by DEDCTA
4.4.4. HPLC Determination of Metabolites
4.5. Statistics
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
2-A-4,6-DCP | 2-Amino-4,6-dichlorophenol |
3,5-DCA | 3,5-Dichloroaniline |
3,5-DCAA | 3,5-Dichloroacetanilide |
3,5-DCNB | 3,5-Dichloronitrobenzene |
3,5-DCPHA | 3,5-Dichlorophenylhydroxylamine |
4-A-2,6-DCP | 4-Amino-2,6-dichlorophenol |
ASC | Ascorbate |
CYP | Cytochrome P450 |
DEDTCA | Diethyldithiocarbamate |
DMSO | Dimethyl sulfoxide |
FMO | Flavin-containing monooxygenase |
GSH | Glutathione |
IKC | Isolated kidney cells |
LDH | Lactate dehydrogenase |
NAC | N-Acetyl-L-cysteine |
PiBx | Piperonyl butoxide |
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Compound | Retention Time (Min) | Limit of Detection (µg) | Extraction Efficiency (%) |
---|---|---|---|
4-A-2,6-DCP | 3.8 | 0.018 | 75.8 |
2-A-4,6-DCP | 13.8 | 0.018 | 97.8 |
3,5-DCPHA | 24.6 | 17.8 | 0.0 * |
3,5-DCA | 29.4 | 0.002 | 89.5 |
3,5-DCAA | 42.6 | 0.002 | 96.1 |
3,5-DCNB | 57.7 | 0.002 | 79.7 |
Compound | 0.5 mM | 1.0 mM | ||
---|---|---|---|---|
Percentage of Dose (%) | ||||
Media | Cells | Media | Cells | |
4-A-2,6-DCP | ND | ND | ND | ND |
2-A-4,6-DCP | ND | ND | ND | ND |
3,5-DCPHA | ND | ND | ND | ND |
3,5-DCA | 89.31 ± 6.32 | 6.82 ± 3.33 | 87.13 ± 4.56 | 13.19 ± 2.12 |
3,5-DCAA | 1.86 ± 0.42 | 0.07 ^ | 0.56 ± 0.07 | ND |
3,5-DCNB | 2.80 ^ | 0.41 ^ | 1.18 ± 0.57 # | 0.34 ± 0.19 # |
Total | 93.97 ± 5.52 | 7.30 ± 3.32 | 88.83 ± 5.14 | 13.53 ± 5.14 |
Compound | −DEDTCA | +DEDTCA | ||
---|---|---|---|---|
Percent of Dose (%) | ||||
Media | Cells | Media | Cells | |
3,5-DCA | 89.43 ± 5.66 | 16.01 ± 2.02 | 91.14 ± 3.04 | 14.67 ± 1.78 |
3,5-DCAA | 0.50 ± 0.03 | 0.02 ^ | 0.30 ± 0.05 | ND |
3,5-DCNB | 0.31 ± 0.19 # | 0.13 ^ | 0.09 ^ | 0.06 ^ |
Pretreatment | Concentration | Pretreatment Time | Mechanism of Action |
---|---|---|---|
(mM) | (Min) | or Enzyme Inhibited | |
α-Tocopherol | 1 | 5 | Antioxidant |
Ascorbate | 1.0 or 2.0 | 5 | Antioxidant |
Glutathione | 1 | 30 | Antioxidant |
N-Acetyl-L-Cysteine | 2 | 30 | Antioxidant |
Piperonyl Butoxide | 1 | 15 | CYP General |
Metyrapone | 1 | 5 | CYP General |
n-Octylamine | 2 | 5 | FMO |
Methimazole | 1 | 30 | FMO |
Indomethacin | 1 | 15 | Cyclooxygenaase |
Mercaptosuccinate | 0.1 | 15 | Peroxidase |
DEDTCA | 0.1 | 30 | CYP2C > CYP2E |
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Rankin, G.O.; Racine, C.R.; Valentovic, M.A.; Anestis, D.K. Nephrotoxic Potential of Putative 3,5-Dichloroaniline (3,5-DCA) Metabolites and Biotransformation of 3,5-DCA in Isolated Kidney Cells from Fischer 344 Rats. Int. J. Mol. Sci. 2021, 22, 292. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010292
Rankin GO, Racine CR, Valentovic MA, Anestis DK. Nephrotoxic Potential of Putative 3,5-Dichloroaniline (3,5-DCA) Metabolites and Biotransformation of 3,5-DCA in Isolated Kidney Cells from Fischer 344 Rats. International Journal of Molecular Sciences. 2021; 22(1):292. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010292
Chicago/Turabian StyleRankin, Gary O., Christopher R. Racine, Monica A. Valentovic, and Dianne K. Anestis. 2021. "Nephrotoxic Potential of Putative 3,5-Dichloroaniline (3,5-DCA) Metabolites and Biotransformation of 3,5-DCA in Isolated Kidney Cells from Fischer 344 Rats" International Journal of Molecular Sciences 22, no. 1: 292. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010292