Insights into the cellular pharmacokinetics and pharmacodynamics of thiopurine antimetabolites in a model of human intestinal cells.

Thiopurines, immunomodulating drugs used in the management of different chronic autoimmune conditions and as anti-leukemic agents, may exert in some cases gastrointestinal toxicity. Moreover, since these agents are administered orally, they are absorbed across the gastrointestinal tract epithelium. On these premises, cellular and molecular events occurring in intestinal cells may be important to understand thiopurine effects. However, quantitative information on the biotransformation of thiopurines in intestinal tissues is still limited. To shed light on biotransformation processes specific of the intestinal tissue, in this study thiopurine metabolites concentrations were analyzed by an in vitro model of human healthy colon, the HCEC cell line, upon exposure to cytotoxic concentrations of azathioprine or mercaptopurine; the investigation was carried out using an innovative mass spectrometry method, that allowed the simultaneous quantification of 11 mono-, di-, and triphosphate thionucleotides. Among the 11 metabolites evaluated, TIMP, TGMP, TGDP, TGTP, MeTIMP, MeTIDP and MeTITP were detectable in HCEC cells treated with azathioprine or mercaptopurine, considering two different incubation times before the addition of the drugs (4 and 48 h). Different associations between metabolites concentrations and cytotoxicity were detected. In particular, the cytotoxicity was dependent on the TGMP, TGDP, TGTP and MeTITP concentrations after the 4 h incubation before the addition of thiopurines. This may be an indication that, to study the association between thiopurine metabolite concentrations and the cytotoxicity activity in vitro, short growth times before treatment should be used. Moreover, for the first time our findings highlight the strong correlation between cytotoxicity and thiopurine pharmacokinetics in HCEC intestinal cells in vitro suggesting that these cells could be a suitable in vitro model for studying thiopurine intestinal cytotoxicity.

Multigenerations assessment of dietary nucleotides consumption in weaned rats.

With the multifaceted activities of nucleotides, there is a history of safe consumption of dietary nucleotides (NTs) in the human diet. This study investigated the multigenerations cumulative toxicity on rats' development after weaning. Weaning rats (F0) were fed with NTs at the dosage of 0.01, 0.04, 0.16, 0.64, and 1.28% (wt/wt) for 90 days and then mated for 1:1 pattern. The offspring was F1. F1 rats were fed with NTs for 90 days after weaning. Afterwards, F1 go on to the second reproductive part. We repeated the above process, until F3 rats were born. We observed the weight, food consumption in the whole experiment, and detected the blood indicators and organ pathology at the terminal. No abnormal reaction, behavior disorder, and organ pathology related to toxic symptom were observed in NTs-treated groups. Weight gain and diet utilization ratio of males in each NTs group had significant increase after weaning (p < 0.05). With the exception of decrease in uric acid (p < 0.05) of NTs male, there were no differences between the control and NTs groups in blood indicators. NTs could promote early growth and development of rats after weaning, especially in males.

Repair of the major lesion resulting from C5'-oxidation of DNA.

Oxidation of the C5'-position of DNA results in direct strand scission. The 3'-fragments produced contain DNA lesions at their 5'-termini. The major DNA lesion contains an aldehyde at its C5'-position, but its nucleobase is unmodified. Excision of the lesion formed from oxidation of thymidine (T-al) is achieved by strand displacement synthesis by DNA polymerase ß (Pol ß) in the presence or absence of flap endonuclease 1 (FEN1). Pol ß displaces T-al and thymidine with comparable efficiency, but less so than a chemically stabilized abasic site analogue (F). FEN1 cleaves the flaps produced during strand displacement synthesis that are two nucleotides or longer. A ternary complex containing T-al is also a substrate for the bacterial UvrABC nucleotide excision repair system. The sites of strand scission are identical in ternary complexes containing T-al, thymidine, or F. UvrABC incision efficiency of these ternary complexes is comparable as well but significantly slower than a duplex substrate containing a bulky substituted thymidine. However, cleavage occurs only on the 5'-fragment and does not remove the lesion. These data suggest that unlike many lesions the redundant nature of base excision and nucleotide excision repair systems does not provide a means for removing the major damage product produced by agents that oxidize the C5'-position. This may contribute to the high cytotoxicity of drugs that oxidize the C5'-position in DNA.

Distribution of purine nucleotides in uremic fluids and tissues.

There are almost 100 different substances called uremic toxins. In this study, we analyze all findings concerning the new family of uremic compounds--nicotinamide end products: N-methyl-2-pyridone-5-carboxamide (Met2PY), N-methyl-4-pyridone-5-carboxamide, newly described 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) and 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside triphosphate (4PYTP). After few years of studies, we have found that these substances have higher plasma concentration in patients with chronic renal failure (CRF) in comparison with the healthy population. We noted a 40-fold increase in plasma 4PYR concentration in patients with CRF. This increment correlates significantly with the decline of kidney function measured as an increase of serum creatinine concentration and decrease of estimated glomerular filtration rate. Tested compounds are present and measurable in physiological fluids and tissues. We found higher saliva Met2PY concentration in patients with CRF in comparison with controls. Saliva Met2PY correlated negatively with estimated glomerular filtration rate and positively with serum creatinine concentration. One-third of studied group had higher concentration of Met2PY in the saliva than in plasma, and this segment of patients may be called as "good excretors." In rats with experimental CRF, we found that both Met2PY and N-methyl-4-pyridone-5-carboxamide accumulated in selected tissues. We also demonstrated formation of 4PYTP in intact human erythrocytes during incubation with the precursor 4PYR. Incubation with 4PYR leads to lowering concentration of adenosine-5'-triphosphate. 4PYTP formation may be a way to remove 4PYR from the circulation and save adenosine-5'-triphosphate depletion. Summarizing, end products of the nicotinamide family are members of uremic toxins; however, exact pathophysiological role of these compounds in the development of uremic syndrome needs further studies.

MTH1, an oxidized purine nucleoside triphosphatase, prevents the cytotoxicity and neurotoxicity of oxidized purine nucleotides.

In human and rodent cells, MTH1, an oxidized purine nucleoside triphosphatase, efficiently hydrolyzes oxidized dGTP, GTP, dATP and ATP such as 2'-deoxy-8-oxoguanosine triphosphate (8-oxo-dGTP) and 2'-deoxy-2-hydroxyadenosine triphosphate (2-OH-dATP) in nucleotide pools, thus avoiding their incorporation into DNA or RNA. MTH1 is expressed in postmitotic neurons as well as in proliferative tissues, and it is localized both in the mitochondria and nucleus, thus suggesting that MTH1 plays an important role in the prevention of the mutagenicity and cytotoxicity of such oxidized purines as 8-oxoG which are known to accumulate in the cellular genome. Our recent studies with MTH1-deficient mice or cells revealed that MTH1 efficiently minimizes accumulation of 8-oxoG in both nuclear and mitochondrial DNA in the mouse brain as well as in cultured cells, thus contributing to the protection of the brain from oxidative stress.

Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.