10:45 AM - 11:00 AM
[MZZ51-07] Genome-wide DNA methylation analysis of dogs with high lead exposure
living near a lead mining area in Kabwe, Zambia
Keywords:DNA methylation, Lead
Variability in gene expression can result from epigenetic changes. The results of epigenetic changes can be as significant as DNA sequence mutations. However, epigenetic marks are reversible and responsive to the environment, unlike DNA mutations. DNA methylation is an epigenetic mechanism wherein cytosine is converted to 5-methylcytosine at cytosine-guanine (CpG) dinucleotides. This biological process causes structural changes that affect DNA–protein interactions. High levels of DNA methylation are generally inversely correlated with gene expression.
As environmental pollutants, lead (Pb) exposure produces several disease symptoms, such as anemia and nephropathy, and can even cause fatalities among humans and companion animals. Even at low levels, Pb exposure can impair pediatric neurodevelopment, and reduced intelligence quotient (IQ) has been reported There is a growing body of research on the role of molecular factors in the etiology of diseases caused by heavy metals. Exposure to metals may contribute to epigenetic modifications as studies have shown that cells exposed to environmental metals display de novo hypermethylation. In addition, global methylation is thought to indicate changes in environment-based responses and chemical exposure, thus suggesting a crucial role of DNA methylation in the mechanisms of diseases caused by environmental pollutants.
Humans and animals are exposed to high levels of Pb in Kabwe's lead-zinc mine in Zambia. Our previous study found that higher levels of Pb in the blood of Kabwe's domestic dogs, which exceeded the toxic level of 40 μg/dL. Dogs are sentinels for toxic substances and share risk factors with their owners. Thus, dogs could be useful in the determination of the extent of Pb exposure among Kabwe residents. We hypothesized that high Pb exposure in dogs (Pb over 10 μg/dL) in Kabwe produce prominent changes in the DNA methylation of key metabolic and neuronal genes.
Four mL of blood was collected from 125 dogs (captive and wild) in Kabwe. Blood lead concentrations were measured by inductively coupled plasma mass spectrometry, and the animals were classified into two groups: a high exposure group (top 10 animals; over 30 ug/dl) and a low exposure group (bottom 10 animals; below 10 ug/dl). DNA extracted from blood was used for genome-wide DNA methylation analysis. The extracted DNA was cleaved by sequential digestion with the DNA methylation-sensitive/insensitive restriction enzymes, SmaI and XmaI. After adapter ligation, fragments of the target size (250-500 bp) were purified and amplified by PCR reaction to prepare libraries for next-generation sequencing using Illumina HiSeq3000, which produced DNA methylation data for approximately 100,000 CpG sites in the canine genome for each sample.
First, correlation analysis with all 20 samples showed two major clusters between the high and low exposure groups to lead except for a few samples. Next, we analyzed 70,211 CpG sites with at least 20 reads (5% difference can be analyzed) and found 827 differentially methylated CpG sites between the high and low exposure groups, most of which had higher DNA methylation levels in the high-exposure group. In addition, genes involved in neurogenesis, such as the NGF gene, were found near the differentially methylated CpG sites. DNA methylation levels of these differentially methylated genes were also validated using blood samples from another 20 dogs.
These results suggest that epigenetic changes, especially changes in DNA methylation, are involved in the mechanism of neurotoxic lead poisoning. Similar changes could be occurred in humans and other animals, and if so, it would be expected to establish biomarkers for lead poisoning as well as novel treatment strategy that target epigenetics in the future.
As environmental pollutants, lead (Pb) exposure produces several disease symptoms, such as anemia and nephropathy, and can even cause fatalities among humans and companion animals. Even at low levels, Pb exposure can impair pediatric neurodevelopment, and reduced intelligence quotient (IQ) has been reported There is a growing body of research on the role of molecular factors in the etiology of diseases caused by heavy metals. Exposure to metals may contribute to epigenetic modifications as studies have shown that cells exposed to environmental metals display de novo hypermethylation. In addition, global methylation is thought to indicate changes in environment-based responses and chemical exposure, thus suggesting a crucial role of DNA methylation in the mechanisms of diseases caused by environmental pollutants.
Humans and animals are exposed to high levels of Pb in Kabwe's lead-zinc mine in Zambia. Our previous study found that higher levels of Pb in the blood of Kabwe's domestic dogs, which exceeded the toxic level of 40 μg/dL. Dogs are sentinels for toxic substances and share risk factors with their owners. Thus, dogs could be useful in the determination of the extent of Pb exposure among Kabwe residents. We hypothesized that high Pb exposure in dogs (Pb over 10 μg/dL) in Kabwe produce prominent changes in the DNA methylation of key metabolic and neuronal genes.
Four mL of blood was collected from 125 dogs (captive and wild) in Kabwe. Blood lead concentrations were measured by inductively coupled plasma mass spectrometry, and the animals were classified into two groups: a high exposure group (top 10 animals; over 30 ug/dl) and a low exposure group (bottom 10 animals; below 10 ug/dl). DNA extracted from blood was used for genome-wide DNA methylation analysis. The extracted DNA was cleaved by sequential digestion with the DNA methylation-sensitive/insensitive restriction enzymes, SmaI and XmaI. After adapter ligation, fragments of the target size (250-500 bp) were purified and amplified by PCR reaction to prepare libraries for next-generation sequencing using Illumina HiSeq3000, which produced DNA methylation data for approximately 100,000 CpG sites in the canine genome for each sample.
First, correlation analysis with all 20 samples showed two major clusters between the high and low exposure groups to lead except for a few samples. Next, we analyzed 70,211 CpG sites with at least 20 reads (5% difference can be analyzed) and found 827 differentially methylated CpG sites between the high and low exposure groups, most of which had higher DNA methylation levels in the high-exposure group. In addition, genes involved in neurogenesis, such as the NGF gene, were found near the differentially methylated CpG sites. DNA methylation levels of these differentially methylated genes were also validated using blood samples from another 20 dogs.
These results suggest that epigenetic changes, especially changes in DNA methylation, are involved in the mechanism of neurotoxic lead poisoning. Similar changes could be occurred in humans and other animals, and if so, it would be expected to establish biomarkers for lead poisoning as well as novel treatment strategy that target epigenetics in the future.