Mechanism of metolachlor action due to alterations in cell cycle progression.

Metolachlor, a commonly used herbicide in the Midwestern USA, functions by inhibiting chlorophyll and protein synthesis in target plants. Herbicide exposure has led to detrimental effects in several organisms, affecting their growth and behavior; however, its mechanism of action in nontarget organisms is not yet clear. The EPA does not currently have enforceable regulations for maximal limits allowed in drinking water. Previous growth studies from our lab have demonstrated that increasing metolachlor concentrations and increasing time of exposure results in decreased growth of liver cells. The objective of this study was to elucidate a mechanism for this decrease of HepG2 cell growth after herbicide exposure. Results show that metolachlor at environmentally relevant levels (50-100 ppb) that previously led to decreased cell number does not lead to cell death by either necrosis or apoptosis. However, it was demonstrated that the levels of the retinoblastoma protein including two of its hyperphosphorylated forms are decreased in metolachlor exposed cells possibly leading to cell cycle arrest. The levels of another protein involved in cell cycle progression, p53, a mediator in the DNA damage response of cells, was not significantly altered except at the highest level of metolachlor (1,000 ppb) and after a 72-h exposure. These results suggest that the decrease in cell number after low-level metolachlor exposure is most likely due to an alteration in the cell cycle and not due to cell death in human liver cells.

Cellular effects of metolachlor exposure on human liver (HepG2) cells.

Metolachlor is one of the most commonly used herbicides in the United States. Protein synthesis is inhibited when roots and shoots of susceptible plants absorb this synthetic herbicide. While quite effective in killing weeds, several studies have shown that exposure to metolachlor results in decreased cell proliferation, growth and reproductive ability of non-target organisms. However, the mode of metolachlor action in non-target organisms has not yet been elucidated. The current study assessed effects of metolachlor exposure on immortalized human liver (HepG2) cells. Results from cell proliferation assays showed that a 72-h exposure to 50 parts per billion (ppb) metolachlor significantly inhibited growth of these cells compared to untreated controls while a decrease in the cell division rate required exposure to 500 ppb metolachlor for 48 h. Flow cytometry analysis of cell cycle distribution revealed that 500 ppb metolachlor treatment resulted in fewer HepG2 cells in G2/M phase after 72 h. Real-time PCR analysis showed a significant decrease in the abundance of the cyclin A transcripts after 12h in cells exposed to 300 ppb metolachlor. These results suggest metolachlor may affect progression through the S phase of the cell cycle and entrance into the G2 phase.

Atrazine exposure leads to altered growth of HepG2 cells.

Atrazine is one of the most commonly used herbicides in the United States. While effective on target plants, it has been associated with harmful health effects in non-target organisms such as fish, amphibians and mammals. In this study, growth effects on human liver cells were determined after exposure to increasing concentrations of this herbicide. Growth of immortalized human hepatoma HepG2 cells was inhibited by atrazine concentrations of 625 ppb after 72 h exposure and flow cytometry analysis demonstrated HepG2 cells exposed to 100 ppb atrazine accumulated in S phase after 48 h compared to untreated cells. Expression of cell cycle specific cyclin proteins was altered after atrazine exposure with cyclin E levels significantly decreased after a 24h exposure and cyclin B levels decreased after 48 h. This study demonstrates that relatively low levels of atrazine exposure can affect growth and lead to disruptions in the cell cycle regulation of immortalized human liver cells.