Blood metallothionein transcript as a biomarker for metal sensitivity: low blood metallothionein transcripts in arsenicosis patients from Guizhou, China

Abstract

Background: Because metallothionein (MT) is a metal-binding protein that protects against metal intoxication, it could be a biomarker for individual sensitivity to metal toxicity.

Objective: We assessed the use of bloodborne MT transcript as a reflection of tissue MT levels and examined the potential role of MT in arsenic toxicity in an environmentally exposed human population.

Method: Rodents were treated with zinc or nonmetallic MT inducers for 4 days, and the blood and tissues were collected for MT transcript analysis by real-time reverse transcriptase-polymerase chain reaction and MT protein determination by the cadmium-hemoglobin assay. Blood and buccal cell samples were collected from arsenicosis patients and healthy subjects residing in Guizhou, China, and total RNA was isolated for MT transcript analysis.

Results: There was a positive correlation between blood MT-1 and MT-2 transcripts and corresponding hepatic or renal MT transcript levels in rats and mice. Furthermore, there was a positive correlation between blood MT-1 and MT-2 transcript and tissue MT protein levels in these animals. A positive correlation also occurred between human blood MT and buccal cell MT transcript levels. MT-1A and MT-2A were the major isoform transcripts in human blood and buccal cells, and significantly lower MT levels were seen in arsenicosis patients compared with healthy subjects.

Conclusions: Blood MT transcript appears to be a useful biomarker of tissue MT levels. Arsenicosis patients in Guizhou show significantly lower MT transcript levels in blood, which may have predisposed this population to arsenic intoxication.

Figure 1

Correlation analysis of blood transcripts with tissue MT transcripts in rats. (A) Correlation of blood MT-1 transcript with hepatic MT-1 transcript. (B) Correlation of blood MT-1 transcript with renal MT-1 transcript. (C) Correlation of blood MT-2 transcript with hepatic MT-2 transcript. (D) Correlation of blood MT-2 transcript with renal MT-2 transcript. Rats (n = 32) were treated with various MT inducers as detailed in “Materials and Methods,” and MT transcript levels were quantified by real-time RT-PCR.

Figure 2

Correlation analysis of (A) hepatic MT protein with hepatic MT-1 mRNA levels; (B), renal MT protein with renal MT-1 mRNA levels; (C) hepatic MT protein with blood MT-1 mRNA levels; and (D) renal MT protein with blood MT-1 mRNA levels. Rats (n = 32) were treated with various MT inducers as detailed in “Materials and Methods”; MT transcript levels were quantified by real-time RT-PCR, and MT protein was determined by the cadmium–hemoglobin assay.

Figure 3

The expression of human MT isoforms in blood cells of arsenic-exposed patients (n = 48) and healthy subjects (n = 48) in Guizhou, China. Total RNA was extracted, purified, reverse-transcripted, and subjected to SYBR Green real-time quantitative RT-PCR with MT isoform primers. Data are mean ± SE. *Significantly different from healthy controls at p < 0.05.

Figure 4

The sex difference in human MT-1A and MT-2A expression in blood cells of arsenicosis patients (26 males and 22 females) and healthy subjects (23 males and 25 females) in Guizhou, China. Total RNA was extracted, purified, reverse-transcripted, and subjected to SYBR Green real-time quantitative RT-PCR with MT isoform primers. Data are mean ± SE.

Figure 5

The expression of human MT isoforms in buccal cells of arsenic-exposed patients (n = 44) and healthy subjects (n = 12) in Guizhou, China. Total RNA was extracted, purified, reverse-transcripted, and subjected to SYBR Green real-time quantitative RT-PCR with MT isoform primers. Data are mean ± SE. *Significantly different from controls at p < 0.05.

Figure 6

Correlation analysis of MT-1A (A) and MT-2A (B) expression between blood cells and buccal cells of arsenicosis patients from Guizhou, China (n = 36). Sample collection and MT determination by real-time RT-PCR are described in detail in “Materials and Methods.”