Indiana University experiments link cadmium toxicity to methylation pattern

Daphnia pulex, the common water flea, has been extensively studied to help science understand human disease processes. Daphnia’s short reproductive cycle makes it ideal for studies of genetic changes over generations. It was the first crustacean to have its full genome sequenced. 

New experiments at Indiana University have made use of Daphnia to try to better understand the toxic effects of cadmium, a metal present in coal, some phosphate fertilizers, and, until recently, in rechargeable batteries. 

The experiments suggest that a complete loss of hydroxymethylation of cytosine, one of the four nucleobases that make up the genetic code, could be responsible for toxic effects produced by cadmium. 

Hydroxymethylation is a type of modification of DNA in which one of the hydrogen atoms or methyl (CH₃) groups in the cytosine molecule is replaced by a hydroxymethyl (CH₂OH) group. 

“We’re suggesting that cadmium is eliminating hydroxymethylation by interfering with the proteins which are responsible for forming hydroxymethylcytosine from methylcytosine,” said Nathan Keith, lead author of the paper reporting on the experiment, to Current Science Daily.

The paper appears Oct. 8 in the peer-reviewed journal Environmental Health Perspectives, co-authored by a six-member team led by pioneering toxicologist and evolutionary biologist Joseph Shaw.

Mutation accumulation

The experiments relied on a relatively new laboratory technique known as mutation accumulation. A rapidly reproducing organism, such as the water flea, is propagated under controlled conditions in the laboratory. Then, part of the offspring is exposed to a suspected environmental toxin, while a control sample is not. 

The purpose of such experiments is to mimic the changes that are occurring in nature, while being able to study the changes in the laboratory. Dr. Michael Lynch, an evolutionary biologist who pioneered the mutation accumulation method in the 1990s when at Indiana, was an author and adviser to the project. 

This was "the first such study, to our knowledge,” to look at how cadmium changes the genome-wide mutation rate, according to Keith. 

An important feature of the experiment was the use of clonal propagation of the Daphnia genotype. In this case a single female Daphnia from Buck Lake in Dorset, Ontario, was the origin of all the asexually produced offspring studied in the experiment. If sibling mating were used, as occurs in nature, it would increase the occurrence of recessive genes containing potentially lethal mutations that would lead to loss of some of the lines. 

Randomly selected offspring were exposed to levels of cadmium found in contaminated areas, while a control sample was propagated in water containing an average background level of cadmium. 

The study followed the broods for an average of 55 generations, before subjecting their sub-lines to deep coverage, whole genome sequencing capable of analyzing about 50 million sites on each genome. 

Surprising results

The results showed that cadmium-exposure increases one class of mutations in some areas of the genome but decreases a different class of mutations in other areas. 

The higher rate of mutation in the cadmium-exposed samples occurred in the intergenic regions, the part of the DNA strand that does not code for proteins. Typically, mutations here are thought to be less damaging. These mutations were of the A : T –> G : C type (where the letters A, T, G, and C refer to adenine, thymine, guanine, and cytosine, the four nucleobases found in DNA).

The lower rate of mutation in the cadmium-exposed samples occurred in the exons, the protein-coding regions of the genome, where mutations are typically more damaging. The less-frequent mutations were of the C : G –> G : C type. 

“We were very surprised by that,” Keith said, “and it hasn’t been reported before, either.” 

Hydroxymethylation

A closer examination of the mutations in the exon regions showed that the reduction in the C : G –> G : C mutation was associated with a complete loss of hydroxymethylation of the cytosine (C) in the DNA. This, too, was unexpected and its significance is yet to be determined. 

Keith suspects the decrease in exon mutations is “linked to changes in an epigenetic process.” Epigenetic changes are those that do not change the genetic code but may produce significant and sometimes heritable changes in an organism. 

His best guess about the loss of hydroxymethylation in presence of cadmium is that the mutation interferes with a type of protein, known as TET, which converts methylcytosine to hydroxymethylcytosine. 

Much, however, is still to be learned about this theory. As Keith points out, the hydroxymethylation of cytosine was only discovered in 2009. 

“I think the next step is to determine what happens in the germline when you knock hydroxymethylation down––what is it controlling?” Keith said. “That’s a very interesting avenue of future study that I would love to look into.”

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Nathan Keith et al. Genome-wide analysis of cadmium-induced, germline mutations in a long-term Daphnia pulex mutation-accumulation experiment. Environmental Health Perspectives (October 2021). DOI: https://doi.org/10.1289/EHP8932