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Earthworms (Lumbricus terrestris) acclimated at 2 degrees C above their habitat temperature (10-12 degrees C) showed about 5% increase in basal rate of oxygen consumption, which increased to about 38% in 14-16 degrees C- and 40% in 16-18 degrees C-, but decreased by 84% in 20-22 degrees C-acclimated worms. Temperature also increased the blood hemoglobin (Hb) concentration, which decreased slightly in 20-22 degrees C-acclimated worms. The worms acclimated at 20-22 degrees C showed their blood to be hypovolemic than that of 10-12 degrees C worms indicating dehydration. Pre-exposure of 10-14 degrees C-acclimated worms to sublethal concentrations of zinc, copper, and lead did not significantly affect the rate of respiration. However, at higher temperatures all these metals inhibited oxygen consumption; zinc, lead, and cadmium by approximately 11% and copper by approximately 18% of that at 14-16 degrees C. At 20-22 degrees C, the respiration was further inhibited, 36% by copper, 18% by cadmium, and approximately 10% by lead and zinc. Copper, lead, and zinc decreased the temperature-enhanced increase in blood Hb concentration at all temperatures. In 20-22 degrees C-acclimated worms heavy metal exposure slightly lowered the oxygen affinity of Hb as well as caused shifts in carbon monoxide difference spectra. The acute toxicity of these metals was not affected by a 2 degrees C rise in acclimation temperature but increased by 17% (lead), 33% (copper), and 5% (zinc) in 14-16 degrees C- and by 40% (lead), 149% (copper), and 132% (zinc) in 20-22 degrees C-acclimated worms. The increase in toxicity of metals caused by high temperatures may be due to limiting the scope of aerobic metabolism (oxygen extraction, transport, and utilization) via quantitative and qualitative effects on Hb. This terrestrial species appears to be tolerant of slight increases in habitat temperature, such as that expected with current global climate change.

BackgroundThe FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage.MethodsWe performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot.ResultsComputational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA.ConclusionsOur results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer.

Amphetamine, a CYP2D6 substrate, is widely used by truck drivers, and the extent to which different people metabolize the drug has only been determined in an isolated or reduced number of samples. A gas chromatography-mass spectrometry method is implemented to simultaneously determine amphetamine, methamphetamine, and hydroxyamphetamine in the urine of drug users. This method is a useful contribution to a well-established field. The main improvements are the use of liquid-liquid extraction, the trapping of the amphetamines as their hydrochloride salt, as a solution to the volatility of these analytes, and its application to assess the CYP2D6 metabolic phenotype of amphetamine users, which is innovative. Calibration curves ranged from 125 to 1000 ng/mL and had an r(2) greater than 0.99. The validation data (precision, accuracy, and recovery) shows the reproducibility and selectiveness of the method. The method is applied to determine the metabolic ratio (MR) in 121 urine specimens of federal highway drivers who underwent random mandatory roadside testing for drugs. The statistical analysis of the MR shows the presence of three different groups, which according to the established groups for CYP2D6 and the amount of the drug metabolized, are classified into extensive metabolizers (EM), intermediate metabolizers (IM), and poor metabolizers (PM). The biological consequences of these differences in amphetamine metabolism, such as impaired driving, a risk to develop Parkinson's disease, or an addiction, need to be further studied.

Arsenic is a common environmental toxicant and epidemiological studies associate arsenic exposure with various pathologic disorders and several types of cancer. Skin cancers are the most common arsenic-induced neoplasias and the prevalence of skin lesions has been reported to be significantly elevated in individuals exposed to arsenic via drinking water in Mexico. Being lymphocytes the main cells used for human monitoring, we evaluated the expression of p53 protein in the lymphocytes from 44 healthy individuals and 19 samples from individuals living in a chronic arsenicism endemic region. Of the latter group, 12 individuals had non-melanoma skin cancer and 9 of them expressed p53 in the circulating lymphocytes, whereas only one of the 7 non-cancer arsenic exposed individuals expressed it. In the healthy non-arsenic exposed group only one from 44 individuals expressed the protein. These results suggest a clear relationship between non-melanoma skin cancer and p53 expression in circulating lymphocytes. p53 expression in circulating lymphocytes should be evaluated as a potential biomarker of effect or susceptibility.