Lead chloride hits some genotypes far harder than others

Lead has been poisoning living things since antiquity, yet its exact mode of action at the cellular level remains surprisingly murky. A new study published in BioRisk has now thrown fresh light on how lead chloride (PbCl2) inflicts damage depending on the genotype, using three model organisms and a battery of microbiological, biochemical, and molecular approaches. The findings reveal that the magnitude of stress response towards PbCl2 is genotype-specific, with one model organism emerging as remarkably sensitive to very low levels of the toxic metal.

The research team, led by scientists investigating the mode of action (MoA) of lead chloride, employed three distinct model systems: the unicellular green alga Chlamydomonas reinhardtii strain 137C (wild type), the yeast Saccharomyces cerevisiae strain D7ts1, and the garden pea Pisum sativum L. cultivar Ran1. Each model was chosen for specific advantages. Chlamydomonas reinhardtii serves as a robust proxy for plant cells in genetic, molecular, physiological, and ecotoxicological studies, with results extrapolatable to a wide variety of plant organisms. Saccharomyces cerevisiae, whose fully sequenced genome shares around 31 percent similarity with the human genome, offers highly conserved stress-response pathways and protein similarity with higher eukaryotes including humans. Pisum sativum L., a classic plant model and one of the most important food items among legume crops, rounds out the trio.

The experimental design covered both short-term and long-term treatments. Cell suspensions of Chlamydomonas and yeast at a density of 1×10^6 cells per millilitre were treated with PbCl2 concentrations ranging from 0.45 to 3.6 mM for two hours. When the extremely low survival of the alga became apparent at this range, researchers added lower concentrations of 0.03, 0.06, 0.11, and 0.22 mM. Pea seeds were treated for 24 and 48 hours to evaluate germination and root length, while pea plants were grown for up to 10 days in contaminated medium at concentrations of 0.45 to 2.7 mM. The highest concentration of 3.6 mM was excluded from long-term experiments because it caused outright plant death.

The results exposed dramatic genotype-related differences in the response to lead treatment. When treated with concentrations in the range of 0.45–3.6 mM, Saccharomyces cerevisiae showed a dose-dependent decrease in cell survival down to 25 percent, while Chlamydomonas reinhardtii dropped to around 10 percent survival across all concentrations tested. Based on survival data, the team calculated that an approximately 30-fold lower dose can cause 50 percent lethality in the alga compared with the yeast. In other words, concentrations that barely trouble the yeast are devastating for the alga, a finding with important implications for how we choose sentinel organisms in environmental monitoring.

Perhaps the most striking discovery concerns DNA damage. Using constant field gel electrophoresis (CFGE), the researchers measured the induction of DNA double-strand breaks (DSBs), the most dangerous form of DNA lesion. For the first time, experimental evidence was obtained that PbCl2 induces DSBs in both Chlamydomonas reinhardtii and Saccharomyces cerevisiae. A statistically significant dose-dependent increase in DSBs was found in the alga at concentrations up to 0.11 mM, corresponding to the LD80. Treatment with 0.45 mM resulted in significant DNA degradation. Interestingly, when concentrations corresponding to equivalent lethality doses were compared across the two organisms, both showed around a 2-fold induction of DSBs at LD80, suggesting a shared underlying mechanism despite the vastly different concentration ranges required.

Crucially, no mutagenic effect was detected in either unicellular model. The mutagenic index in Chlamydomonas was calculated to be less than 2.5, indicating no mutagenic capacity, while mitotic gene conversion and reverse mutations in yeast were comparable with untreated controls. This provides indirect evidence that PbCl2 toxicity at the tested concentrations is not related to point mutations, impaired cell division, micro-chromosomal aberrations, mitotic gene conversion, or mitotic crossing-over. The damage appears to be clastogenic rather than mutagenic, breaking DNA strands rather than altering their sequence.

The pro-oxidative potential of PbCl2 was confirmed in all model systems, covering a wide range of reactive oxygen species (ROS). In yeast, a dose-dependent increase in superoxide anion levels was measured, with more than 2-fold higher levels at LD80, alongside elevated malondialdehyde (MDA) and depleted glutathione (GSH). The reduction in GSH likely reflects the high affinity of the GSH thiol group to lead, forming Pb-GSH complexes in the cytosol. In Chlamydomonas, a dose-dependent increase in intracellular peroxides (H2O2) was obtained, with more than 3-fold higher levels of both MDA and H2O2 at concentrations causing over 80 percent cell lethality. In pea plants, the most pronounced oxidative stress was observed after 10 days of growth in medium contaminated with 2.7 mM PbCl2, with MDA levels rising to 166 percent and H2O2 to 172 percent of controls.

Correlation analysis strengthened the mechanistic picture. In Chlamydomonas, the decrease in cell survival corresponded strongly with increases in H2O2 and DSBs, and higher H2O2 levels correlated with low levels of all photosynthetic pigments. In yeast, the decrease in cell survival correlated strongly with superoxide anion induction, while DSB increases correlated with MDA elevation and GSH depletion. Based on these relationships, the researchers speculate that Pb-induced oxidative stress, particularly in terms of intracellular peroxides, probably participates in the induction of DSBs, which in turn may partially result in cell death. The study provides new evidence that PbCl2 can affect both the DNA molecule and photosynthetic pigments via the induction of oxidative stress.

The photosynthetic machinery took a significant hit in both plant models. All photosynthetic pigments in Chlamydomonas and Pisum sativum decreased by around 40–60 percent in both experimental schemes, without concentration or time dependence. In the alga, chlorophyll a, chlorophyll b, and carotenoids dropped by around 50–60 percent. Some authors attribute lead’s effect on chlorophyll to its ability to replace magnesium in the chlorophyll ring, while others propose that Pb-induced disturbance of photosynthesis in microalgae is a major cause of cell death. In this study, however, no statistically significant correlation was obtained between pigment levels and cell survival, pointing instead to H2O2-mediated growth inhibition as the more likely pathway.

The toxicity of PbCl2 on pea plants proved surprisingly mild. Around 10 percent inhibition of seed germination and around 20 percent root length reduction were measured at concentrations equal to or higher than 1.8 mM. While statistically significant, these effects can hardly be regarded as significant from a biological point of view, confirming previous observations that growth-related parameters are not the most sensitive endpoints for evaluating lead toxicity. The stronger effect on root length than germination may be due to the low permeability of the seed testa to lead.

The finding that Chlamydomonas reinhardtii is a sensitive test system towards PbCl2 contributes to good strategies for revealing very low levels of contaminants present chronically in main environmental matrices. Very sensitive genotypes are excellent tools for detecting chronic low-level contamination that can result in long-term disturbance of biota. The study’s proposed approach, a battery of test systems with various endpoints, could be considered a promising tool in the ecotoxicological assessment of various xenobiotics, in line with WHO recommendations that multiple test systems and endpoints evaluating different adverse effects at different levels provide the most reliable information about the mode of action of environmental contaminants.

Subject of Research: Genotype differences towards lead chloride harmful action

Article Title: Genotype differences towards lead chloride harmful action

Article References: Todorova, T. I., Parvanova, P. N., Boyadzhiev, K. P., Dimitrov, M. D., & Chankova, S. G. (2023). Genotype differences towards lead chloride harmful action. BioRisk, 20, 37-57. https://doi.org/10.3897/biorisk.20.97598

Image Credits: AI Generated

DOI: 10.3897/biorisk.20.97598

Keywords: Genotype, differences, towards, lead, chloride, harmful, action, scientific research