Tandem mass spectrometry measurements of creatinine in mouse plasma and urine for determining glomerular filtration rate

Endogenous creatinine clearance (Ccr) is widely accepted as an estimate of glomerular filtration rate (GFR), the best overall biomarker of kidney function. However, current common methods of measuring creatinine are not sensitive enough for mouse plasma. Accordingly, we here report a new method of measuring creatinine by liquid chromatography tandem mass spectrometry (LC-MS/MS) using deuterated [2H3]-creatinine as an internal standard. The assay requires 10 microl or less of plasma or urine, and is eight times more sensitive than high-performance liquid chromatography. The reproducibility of the assay of replicates is approximately +/-10%. The plasma creatinine levels of wild type male C57BL/6J mice obtained by LC-MS/MS are 0.076+/-0.002 mg/dl (n=65). To estimate daily urinary creatinine excretion for calculating Ccr, we collected urine from mice housed in metabolic cages, and combined this with washes from the cage internal surfaces. Creatinine in the wash varies from 4 to 67% of the total daily urinary creatinine excretion (typically approximately 400 microg/day). Ccr obtained by LC-MS/MS was 329+/-17 microl/min, which is indistinguishable from GFR measured by using fluorescein isothiocyanate-inulin. The LC-MS/MS method is sensitive, specific, simple, fast, and inexpensive; it is suitable for estimating GFR in conscious mice or other small animals. As it allows repeated measurements in the same animals, it facilitates detection of subtle differences or changes in renal function

Synthesis of chlorinated and non-chlorinated biphenyl-2,3-and 3,4-catechols and their H-2(3) -isotopomers

A synthetic scheme is described for chlorinated biphenyl-2,3- and 3,4-catechols to be used as standards for structural assignment of metabolites and protein adducts of 2,2', 5,5'-tetrachlorobiphenyl in which both rings retain chlorine substituents. The scheme has general applicability to the synthesis of chlorinated biphenyl catechols. Dimethyl catechol ethers are coupled to dichloroaniline via the Cadogan reaction to give a library of isomers, followed by demethylation of the ethers with BBr3 to yield the target catechols. Separation of pure isomers is accomplished by TLC or HPLC prior to or following demethylation, depending on the isomer mixture. [H-2(3)]-Isotopomers are generated using 2,5-dichloroanilined-3 as the starting arylamine in the coupling reaction. The dichloroaniline-d(3) hydrochloride is obtained as the sole product from nitration of p-dichlorobenzene-d(4) followed by Pd/C-catalyzed hydrogenation under strongly acidic conditions. This hydrogenation procedure provides a simple and convenient approach to selective reduction of aryl nitro groups in the presence of halogen ring substituents

Molecular dosimetry of N7-(2-hydroxypropyl)guanine in tissues of F344 rats after inhalation exposure to propylene oxide.

Propylene oxide (PO) is a high-volume chemical intermediate that causes a low incidence of nasal tumors in rodents exposed to high concentrations (≥300 p.p.m.). PO reacts with DNA forming mainly N7-(2-hydroxypropyl)guanine (7-HPG). The exposure-dependent accumulation of 7-HPG in nasal respiratory epithelium (NRE), lung and liver was determined in male F344 rats exposed to PO (0, 5, 25, 50, 300 or 500 p.p.m.) by the inhalation route for 3 or 20 days (6 h/day; 5 days/week). These exposures ranged from low concentrations, such as those potentially occurring in the workplace, to high concentrations that proved to be carcinogenic in rodents. Analysis of 7-HPG in DNA by gas chromatography–high-resolution mass spectrometry (GC–HRMS) showed a linear response in 7-HPG for all three tissues after 3 days of exposure, and for NRE and lung after 20 days of exposure. A slightly sublinear response in 7-HPG was observed in liver after 20 days of exposure. For both exposure periods, the NRE had the highest concentration of 7-HPG, followed by lung and liver. The amount of 7-HPG in NRE was seven and 17 times higher than in lung and liver, respectively, for the 3 day exposures. For the 20 day exposures, the concentration of 7-HPG in NRE was six and 13 times higher than that in lung and liver, respectively, over the concentration range studied. These results demonstrate a much higher extent of DNA alkylation in the target tissue for carcinogenesis, than in non-target tissues. As PO-induced tumor formation was highly sublinear, occurring only at high vapor concentrations, whereas 7-HPG adducts were shown to be linearly dependent on airborne concentration, these results suggest that 7-HPG is not sufficient for PO nasal carcinogenesis and that other factors such as increased cell proliferation may be important in determining the tumor exposure response

Aldehydic DNA lesions induced by catechol estrogens in calf thymus DNA

The primary purpose of this research is to examine the hypothesis that reactive oxygen species generated by estrogen quinonoids are the main source for the formation of aldehydic DNA lesions (ADL) in genomic DNA. ADL induced by quinonoid metabolites of 17beta-estradiol (E-2), e.g. 4-hydroxyestradiol (4-OH-E-2), 2-hydroxyestradiol (2-OH-E-2), estrogen-3,4-quinones (E-2-3,4-Q) and estrogen- 2,3-quinone (E-2-2,3-Q), were investigated in calf thymus DNA (CT-DNA) under physiological conditions. The abasic sites resulting from the spontaneous depurination-depyrimidination of the modified bases and the aldehydic base and sugar lesions resulting from the oxidative damage to deoxyribose moieties in the DNA molecules were measured by an aldehyde reactive probe and were estimated as the number of ADL per 10(6) nucleotides. With the addition of NADPH (100 muM) and Cu(II) (20 muM), nanomolar levels (100 nM) of 4-OH-E-2 and 2-OH-E-2 induced similar to10-fold increases in the number of ADL over control (P<0.001). In parallel, increases in 8-oxoguanine were detected in DNA exposed to 4-OH-E-2 and 2-OH-E-2 (100 nM) plus Cu(II) and NADPH. Further investigation indicated that the ADL induced by estrogen catechols plus Cu(II) and NADPH were causally involved in the formation of hydrogen peroxide and Cu(I). Both E-2-2,3-Q and E-2-3,4-Q alone induced a 2-fold increase in the number of ADL over control (P<0.05) in CT-DNA at high concentrations (1 mM). Neither neutral thermal hydrolysis nor lower ionic strength of the reaction medium induced further increases in the number of ADL in E-2-3,4-Q-modified CT-DNA. Conversely, with the inclusion of Cu(II) and NADPH, both E-2-3,4-Q and E-2-2,3-Q (1 muM) induced parallel formation of DNA single strand breaks and similar to20-fold increases in the number of ADL over control (P < 0.001). The data also demonstrated that the ADL induced by estrogen quinones with and without the presence of Cu(II) and NADPH contain 69 and 78% putrescine-excisable ADL in CT-DNA, respectively. Additionally, results of the ADL cleavage assay indicate that the ADL induced by estrogen quinones plus Cu(II) and NADPH in CT-DNA were predominantly T7 exonuclease-excisable (50%) and exonuclease III- excisable (20%) ADL, whereas the intact ADL, and other ADL accounted for 5 and 25%, respectively. These results suggest that the ADL induced by estrogen quinones in CT-DNA are derived from oxidative events rather than depurination/depyrimidination of labile estrogen quinone-DNA adducts. Overall, our results are at variance with the idea that depurination of estrogen quinone-DNA adducts is the major source for the formation of ADL in genomic DNA. We hypothesize that in addition to DNA adducts and oxidized bases, the ADL induced by estrogen quinonoid-mediated oxidative stress may play a role in estrogen-induced carcinogenicity

Basic or applied, it's the interaction that counts - Toxicology must not be driven by politics: it's science that should drive decision-making

Correspondence - no abstract available

Induction of cytotoxicity, aldehydic DNA lesions, and poly(ADP-ribose) polymerase-1 activation by catechol derivatives of pentachlorophenol in calf thymus DNA and in human breast cancer cells

The purpose of this study was to investigate the degree of chlorination of catechol (CAT) derivatives of pentachlorophenol (PCP) on the induction of cytotoxicity and DNA damaging effects in calf thymus DNA (ct-DNA) and in two human breast carcinoma cell lines. Results indicated that with the addition of the transition metal copper(II), increases in the amount of aldehydic DNA lesions (ADL) were detected in ct-DNA exposed to PCP-derived CATs over the corresponding control. The DNA lesions induced by various degrees of chlorination of PCP-derived CATs decrease in the rank order CAT congruent to 4-chlorocatechol (4-CICAT) > 4,5-dichlorocatechol (4,5-Cl(2)CAT) > 3,4,5-trichlorocatechol (3,4,5-Cl(3)CAT) > tetrachlorocatechol (Cl(4)CAT). In contrast, Cl(4)CAT was the only congeneric form of PCP-derived catechols that induced a significant increase in the number of ADL in human MCF-7 cells, and this only occurred when glutathione was depleted. Pretreatment with copper(I) and iron(II) chelators significantly reduced the formation of ADL in cells exposed to Cl(4)CAT. The data also indicated that the ADL induced by Cl(4)CAT in MCF-7 cells contain similar to70% putrescine excisable ADL. This evidence confirmed that the ADL induced by Cl(4)CAT in MCF-7 cells were derived from oxidative events. In addition, we demonstrated that the depletion of NAD(P)H in human T47D cells exposed to chlorinated CATs decreased in the rank order Cl(4)CAT much greater than 4-CICAT congruent to CAT. The depletion of NAD(P)H induced by Cl(4)CAT in T47D cells was partially blocked by catalase, superoxide dismutase, dimethyl sulfoxide, and copper(I) and iron(II) specific chelators. Additionally, the depletion of NAD(P)H in T47D cells exposed to Cl(4)CAT (1-10 muM) was completely blocked by three types of poly(ADP-ribose) polymerase-1 inhibitors. This evidence suggests that Cl(4)CAT induces an imbalance in DNA repair and the subsequent accumulation of DNA strand breaks in human cultured cells. Overall, these findings indicate that dechlorination may decrease the potentials of chlorinated catechols to induce oxidative DNA lesions and cytotoxic effects in living cells