Using glowing fish to detect harmful pesticides

Birth defects related to chromosomal abnormalities often result from exposure to chemicals early in the mother’s life. But identifying the chemicals at fault presents a serious challenge — akin to solving a hit-and-run case, decades after the fact. Two researchers in the UC Davis School of Biological Sciences are developing a method that can identify harmful chemicals much more quickly, with the help of red-green glowing zebrafish.

Their work could benefit millions of people in California’s Central Valley, who are at high risk of pesticide exposure because they live or work near agricultural production sites. Pesticide exposure can cause acute and long-term health problems in humans, including harm to the reproductive system. This damage often occurs because the chemicals interfere with the delicate stages of fetal development, during which cells that will one day produce sperm or eggs are forming.

Finding generational effects

“You won’t see the effect until these kids are older and trying for their own,” said Sean Burgess, a professor in the Department of Molecular and Cellular Biology. At this point, women may experience infertility or recurrent miscarriages; The children who bear them may be at increased risk of developing Down syndrome or other serious conditions caused by the presence of extra copies of chromosomes.

Burgess is working with Bruce Draper, a professor in the same department, to develop technology that can greatly speed up the screening of chemicals and identify those with long-term reproductive effects faster. The gap between a chemical “hit-and-run” and reproductive consequences is often decades, Burgess said: “We’re basically reducing that time to weeks.”

Standard tests are slow and expensive, because they rely on mice, which must be dissected and examined individually by technicians for the effect of chemicals on reproductive tissue. Burgess and Draper plan to circumvent this cumbersome process by using a newly developed strain of zebrafish (Danio rerio). Native to southern Asia, this species of freshwater fish is popular in home aquariums. It is also frequently used as a model organism to study the early stages of human development.

“Seventy percent of the genes in zebrafish have human counterparts called orthologues,” Draper said. And if you look at the genes involved in oogenesis — the production of female eggs, or egg cells — the percentage is even higher.

Zebrafish are well suited to studying the reproductive effects of chemicals because, unlike mammals, their sex is not determined by their own X or Y chromosomes. Instead, it is determined in part by environmental cues. In captivity, about half of the fish develop into females. But if fish larvae are exposed to chemicals that disrupt egg formation, a higher proportion of them will develop as males. This means that scientists can screen a chemical for reproductive toxicity by exposing a few dozen zebrafish larvae to it—and then wait several weeks to see if their sex ratio skews toward males. Draper and Burgess are developing a strategy to do just that—using transgenic zebrafish that display their sex prominently, through color-coding.

Developed by members of Draper’s lab, these fish carry three genetic changes. First, Sertoli cells, which are found only in the male gonads, produce green fluorescent protein. Second, oocytes (or immature oocytes), which are present only in the female gonad, produce red fluorescent protein. Finally, the fish make less of their natural pigment – which makes their bodies more transparent, and thus the red or green colors of their gonads appear more clearly.

Zebrafish are easier and less expensive to care for than ferrets. Burgess and Draper expect to raise 80 fish fry in each tank, exposing the animals in each one to a chosen chemical between 10 and 20 days after fertilization. One usually has to wait until 90 days after fertilization to visually distinguish between a male and a female zebrafish. But the gonads of the fish must allow this to be done in 40 days.

“We should be able to determine the sex of a group of 80 animals, almost simultaneously, just by taking a picture,” Draper said. Seeing an unusually high percentage of males or females, or seeing bisexual animals with gonads that fluoresce red and green, indicates that the chemical is toxic to the reproductive system.

GloNad Check

Earlier this year, Burgess and Draper began developing a “GloNad” toxicity screening test. The two hope that later this year they can start using it in a pilot trial examining nine of the most popular pesticides in California for reproductive effects.

This initial test could eventually pave the way for wider use of the GloNad assay. There are 90 pesticides currently known to the State of California to cause birth defects or reproductive harm. But these toxicities are likely to be related to a wider range of pesticides and other chemicals, such as bisphenols, that are used to manufacture some plastics.

The real strength of the GloNad assay is that it can be scaled up to test many more chemicals than are currently possible. Both of these chemicals can be tested in large numbers of fish – allowing rare reproductive effects to be detected.

“It’s a way more effective than anything else out there right now,” Draper said. “We have high expectations that this will work.”