Toxicity Assessment of 4-Methyl-1-cyclohexanemethanol and Its Metabolites in Response to a Recent Chemical Spill in West Virginia, USA

Abstract

The large-scale chemical spill on January 9, 2014 from coal processing and cleaning storage tanks of Freedom Industries in Charleston affected the drinking water supply to 300,000 people in Charleston, West Virginia metropolitan, while the short-term and long-term health impacts remain largely unknown and need to be assessed and monitored. There is a lack of publically available toxicological information for the main contaminant 4-methyl-1-cyclohexanemethanol (4-MCHM). Particularly, little is known about 4-MCHM metabolites and their toxicity. This study reports timely and original results of the mechanistic toxicity assessment of 4-MCHM and its metabolites via a newly developed quantitative toxicogenomics approach, employing proteomics analysis in yeast cells and transcriptional analysis in human cells. These results suggested that, although 4-MCHM is considered only moderately toxic based on the previous limited acute toxicity evaluation, 4-MCHM metabolites were likely more toxic than 4-MCHM in both yeast and human cells, with different toxicity profiles and potential mechanisms. In the yeast library, 4-MCHM mainly induced chemical stress related to transmembrane transport and transporter activity, while 4-MCHM metabolites of S9 mainly induced oxidative stress related to antioxidant activity and oxidoreductase activity. With human A549 cells, 4-MCHM mainly induced DNA damage-related biomarkers, which indicates that 4-MCHM is related to genotoxicity due to its DNA damage effect on human cells and therefore warrants further chronic carcinogenesis evaluation.

Figure 1.

Dose responses based on a 24 h cytotoxicity (survival) assay of 4-MCHM and its metabolites (4-MCHM+S9) in yeast (A) and human A549 cells (B). X-axis: chemical concentration (mg/L); Y-axis: percentage of surviving cells compared to vehicle control. Mean ± SD, n = 3.

Figure 2.

Temporal altered protein expression profiles of 148 biomarkers indicative of different stress responses upon exposure to 4-MCHM (A) and 4-MCHM metabolites (4-MCHM+S9) (B) across six-log concentrations. The mean natural log of induction factor (ln I) indicates the magnitude of altered protein expression (represented by a green–black–red color scale at the bottom. Red spectrum colors indicate up-regulation; green spectrum colors indicate down-regulation. Values beyond ±2 are shown as ±2). X-axis top: concentrations for each chemical; X-axis bottom: testing time in minutes; the first data point shown is at 20 min after exposure due to data smoothing with a moving average of every five data points. Y-axis left: clusters of proteins by stress response pathways; Y-axis right: list of proteins (ORFs) tested, with details in Table S1, Supporting Information. n = 3. The stress category is labeled with “*” for those with FDR < 25% for gene set enrichment analysis (the statistical significance of the enrichment score for a single set).

Figure 3.

PCA graphical representations of the three major components of protein expression change profiles for 4-MCHM (red square) and 4-MCHM with S9 (green sphere), based on induction factor (ln I) during a 2 h exposure across six-log concentrations. Samples are color coded according to chemicals. Each square or sphere represents one treatment (a chemical at a given concentration) with symbol size indicating the relative level of concentration.

Figure 4.

Altered gene expression profiles of biomarkers indicative of toxicity and stress responses in human cells, upon a 6 h exposure to 4-MCHM only and 4-MCHM +1% S9, respectively, across three concentrations in human A549 cells. The mean natural log of induction factor (ln I) indicates the magnitude of altered gene expression (represented by a green–black–red color scale at the bottom. Red spectrum colors indicate up-regulation; green spectrum colors indicate down-regulation; values beyond ±2 are shown as ±2, values in Figure 5). X-axis top: concentrations tested; Y-axis left: clusters of genes by stress categories; Y-axis right: list of genes tested, with details of gene descriptions in Table 1. n = 3.

Figure 5.

Gene expression change in A549 cells after a 6 h exposure of 4-MCHM and 4-MCHM metabolites (4-MCHM+S9). RT-qPCR indicated gene alteration by fold change (Y-axis), compared to untreated control; *: I >1 with p < 0.05, n = 3.