Morphofunctional changes in the rat's liver of different ages after L-methionine administration

Background: Literature data on the effect of methionine on functional activity and, especially, on morphological changes in the liver parenchyma in animals of different ages are sporadic, and research results are often ambiguous. Aim: The purpose of this work was to study and compare the morphofunctional changes in the liver of rats of different ages on prolonged administration of L-methionine. Material and Methods: The experiment was performed on 48 male Wistar rats of 3 and 15 months of age. Animals of the experimental group received L-methionine at a dose of 250 mg/kg body weight in addition to the standard diet, daily for 21 days. Histological preparations were prepared from liver tissue by a standard technique. Morphometry was performed on digital images using the computer program «Image J». Succinate dehydrogenase activity and protein concentration were determined in the suspension of hepatocyte mitochondria. Results: It was revealed that 21-day administration of L-methionine to rats led to hypertrophy of the hepatocyte nucleus, an increase in the nuclear-cytoplasmic ratio, the number of binuclear hepatocytes, and the nucleolus in the cell nucleus. The relative area of ​​the sinusoids network increased by 50% in 3-month-old animals. This indicated a better blood filling of the liver parenchyma. The increase in succinate dehydrogenase activity and protein concentration was revealed in the suspension of hepatocyte mitochondria of the experimental rats. This indicated an increase in the mitochondria energy potential and protein-synthetic activity. Conclusions: The administration of prophylactic doses of methionine to healthy rats leads to the appearance of pronounced morphological and functional signs of increased activity of hepatocytes. The severity of this effect has a distinct age-dependent character. In young rats, it is more pronounced than in mature rats. The results of the study are important for practical medicine when using methionine for therapeutic and prophylactic purposes.

Organic Amendments and Elemental Sulfur Stimulate Microbial Biomass and Sulfur Oxidation in Alkaline Subtropical Soils

Sulfur deficiency arising due to intensive cultivation, use of sulfur free fertilizers and reduction in atmospheric sulfur depositions has become a major issue limiting crop production in many parts of the world. Elemental sulfur could be a good source of available S, but its slow oxidation is a problem for its efficient use as a sulfur fertilizer. Main objective of the study was to assess the effect of organic amendments (OA) and elemental sulfur (ES) on microbial activities, sulfur oxidation and availability in soil. A laboratory incubation experiment was carried out for a 56 days period using two sulfur deficient alkaline soils. Organic amendments (OA), i.e., farmyard manure (FYM), poultry litter (PL) and sugarcane filter cake (SF), were applied (1% w/w) with or without elemental sulfur (ES) at 50 mg kg−1. Application of ES alone or in combination with OA significantly increasedCO2-C evolution, microbial biomass, and enzyme activities in the soils, except dehydrogenase activity (DHA) which was not affected by ES application. Combined application of OA and ES had a more pronounced effect on microbial parameters compared to ES or OA applied alone. Ratios of dehydrogenase activity-to-microbial biomass C and arylsulfatase activity-to-microbial biomass C were high in ES+FYM and ES+SF treatments, respectively. Elemental sulfur got sufficiently oxidized resulting in significant improvement in plant available S. Relatively more ES was distributed into C-bonded-S than ester bonded-S. Increase in sulfur availability in ES+OA amended soils was the combined function of sulfur oxidation and mineralization processes through improved microbial activity.

Potential added value of combined DPYD/DPD genotyping and phenotyping to prevent severe toxicity in patients with a DPYD variant and decreased dihydropyrimidine dehydrogenase enzyme activity

Decreased dihydropyrimidine dehydrogenase enzyme activity is associated with severe fluoropyrimidine-associated toxicity. Four clinically relevant variants in the DPYD gene are associated with decreased dihydropyrimidine dehydrogenase activity. However, only ∼25% of DPYD variant carriers show a decreased dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells. Objective To investigate if dihydropyrimidine dehydrogenase phenotyping has added value when combined with DPYD genotyping in predicting fluoropyrimidine-related toxicity. Methods Retrospective cohort study in which treatment and toxicity data were collected of 228 patients genotyped for four DPYD variants and phenotyped using an ex vivo peripheral blood mononuclear cell assay. Results Severe toxicity occurred in 25% of patients with a variant and normal dihydropyrimidine dehydrogenase activity, in 21% of patients without a variant and with decreased dihydropyrimidine dehydrogenase activity, and in 29% of patients without a variant and with normal dihydropyrimidine dehydrogenase activity (controls). The majority of patients with a variant or a decreased dihydropyrimidine dehydrogenase activity received an initial dose reduction (68% and 53% vs 19% in controls) and had a lower mean dose intensity (75% and 81% vs 91% in controls). Fifty percent of patients with a variant and decreased enzyme activity experienced severe toxicity, despite the lowest initial dose and whole treatment dose intensity. They also experienced more grade 4/5 toxicities. Conclusions Our results indicate that a combined genotype–phenotype approach could be useful to identify patients at increased risk for fluoropyrimidine-associated toxicity (e.g. patients with a variant and decreased dihydropyrimidine dehydrogenase activity). Because the group sizes are too small to demonstrate statistically significant differences, this warrants further research in a prospective study in a larger cohort.

Influence of heavy metals on physiological and biochemical parameters of Pseudorasbora parva (Cypriniformes, Cyprinidae)

Anthropogenic load on aquatic ecosystems leads to increased inputs of heavy metals, which can have a toxic effect on aquatic organisms. Some of the most appropriate objects for research are short-cycle fish species. This article considers the results of studies on the adaptive reactions of the stone moroko Pseudorasbora parva (Temminck et Schlegel, 1846) to Mn, Pb, Ni heavy metal ions, which exceeded the reference values in the reservoir by 1.7, 1.5 and 2.0 times, respectively. Changes in morphological parameters of the blood and histocytological pattern of the hepatopancreas of the experimental species under the influence of toxicants, as well as changes in biochemical parameters, were determined. It was noted that the influence of Mn caused pathological changes in the form of poikilocytosis. The morphometric parameters of erythrocytes (the cell area and the nuclear area) reached 67.48 ± 0.67 and 13.97 ± 0.22 µm2 respectively (4.0% and 13.8% less compared to the control). The influence of Ni resulted in an increased number of leukocytes and immature forms of erythrocytes. The area of erythrocytes was 0.9% smaller than that of the control group, and the area of the nucleus was 4.5% smaller than in the control. The effect of Pb as well as Mn consisted in poikilocytosis. The area of red blood cells and nuclei was smaller by 6.5% and 8.3%, respectively, compared to the control. The percentage of white blood cells in fish exposed to Ni and Pb tended to increase. In the experiment with nickel, the percentage of white blood cells was 10.2% of the number of formed elements; in the experiment with lead – 11.3%; with manganese – 6.1%, while in the control, the number of white blood cells compared to the total number of formed elements of fish was only 1.2%. Cytometric studies have revealed that there are significant differences in the shape, size, and location of hepatocytes in different experimental fish. The structural components of the liver for histological specimens were stained differentially with different intensities and different colours. The hepatocytes on histological specimens of the liver of the stone moroko exposed to Ni and Mn ions did not have clearly defined boundaries, there was a large number of destroyed cells, which indicates the toxic effect of these heavy metals. The hepatocytes exposed to Pb had the largest area of cells and nuclei and the highest nuclear-cytoplasmic ratio is typical for hepatocytes under the influence of Mn. The nuclei had the largest size and occupied 12.7% of the internal contents of the cells. It is shown that under the impact of the studied concentrations of Mn, Ni and Pb, lactate dehydrogenase activity increased by 1.22, 1.14 and 1.48 times compared to the control, respectively. In contrast, there was a 3.27-fold decrease in succinate dehydrogenase activity under the impact of Mn. Besides that, the activity of the enzyme decreased by 1.48 and 1.68 times under the action of Ni and Pb. Subsequently, we found an increase in the activity of alkaline phosphatase in muscle tissue by 3.25–3.94 times under the influence of the studied toxicants. Muscle protein levels under the impact of Mn were 1.14 times lower than in the control, the most distinct decrease in protein was found under the impact of Ni (1.53 times). The obtained data of physiological and biochemical reactions of the stone moroko to the influence of heavy metals provide an opportunity to predict changes in the species composition of fish fauna under conditions of excessive toxic pollution of ecosystems.

Catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2)

Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg2+ ion, and (b) the substrate-bound form of MTHFD2. Using homology modeling and simulation studies, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate MgA, while MgA together with Arg233 stabilize the inorganic phosphate (Pi); (ii) Asp168 and three water molecules coordinate MgB, and MgB further stabilizes Pi by forming a hydrogen bond with two oxygens of Pi; (iii) Arg201 directly coordinates the Pi; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of MgA, MgB and Pi. Our computational study at the empirical valence bond level allowed us to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution coupled to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues Ser197/Arg233 are key factors in determining the cofactor (NADP+/NAD+) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer therapy.