APC haploinsufficiency coupled with p53 loss sufficiently induces mucinous cystic neoplasms and invasive pancreatic carcinoma in mice.

Adenomatous polyposis coli (APC), a tumor-suppressor gene critically involved in familial adenomatous polyposis, is integral in Wnt/ß-catenin signaling and is implicated in the development of sporadic tumors of the distal gastrointestinal tract including pancreatic cancer (PC). Here we report for the first time that functional APC is required for the growth and maintenance of pancreatic islets and maturation. Subsequently, a non-Kras mutation-induced premalignancy mouse model was developed; in this model, APC haploinsufficiency coupled with p53 deletion resulted in the development of a distinct type of pancreatic premalignant precursors, mucinous cystic neoplasms (MCNs), exhibiting pathomechanisms identical to those observed in human MCNs, including accumulation of cystic fluid secreted by neoplastic and ovarian-like stromal cells, with 100% penetrance and the presence of hepatic and gastric metastases in >30% of the mice. The major clinical implications of this study suggest targeting the Wnt signaling pathway as a novel strategy for managing MCN.

Effects of methionine-induced hyperhomocysteinemia on endothelium-dependent vasodilation and oxidative status in healthy adults.

BACKGROUND: Homocysteine-mediated endothelial dysfunction has been proposed to occur via oxidative stress mechanisms in humans. However, there is controversy regarding the effects of homocysteine on endothelial function and oxidative status, which may in part result from age discrepancy across the studies. The present study was designed to investigate the aging effect on the relationship between endothelium-dependent vasodilation and oxidative status in methionine-induced hyperhomocysteinemia. METHODS AND RESULTS: Plasma homocysteine, phosphatidylcholine hydroperoxide (PCOOH), P-selectin levels, and brachial artery flow-mediated vasodilation were measured at baseline and 4 hours after an oral methionine load (0.1 g/kg) in 15 younger (21 to 40 years) and 15 older (55 to 70 years) healthy adults. Homocysteine increased from 7.3+/-1.3 micromol/L at baseline to 22.7+/-5.2 micromol/L at 4 hours in younger (P<0.001) and from 7. 4+/-1.4 to 24.3+/-4.5 micromol/L in older adults (P<0.001). PCOOH levels were not significantly different between baseline and 4 hours in both groups (P=0.10 in young; P=0.14 in old). P-selectin, which is expected to increase during oxidative stress, was not changed in older (P=0.08) but decreased in younger adults (P=0.037) at 4 hours. Flow-mediated vasodilation was preserved from 13.1+/-2.1% at baseline to 13.5+/-2.8% at 4 hours in younger (P=0.49) and decreased from 12.8+/-2.4% to 8.5+/-2.8% in older adults (P<0.001). CONCLUSIONS: The present study demonstrates that endothelial dysfunction caused by methionine-induced hyperhomocysteinemia is age-related and is mediated through impaired nitric oxide activity without change of oxidative status. Our data do not support previous hypotheses that endothelial damage by homocysteine is via oxidative stress mechanism in humans.

Increased prevalence of hypertension and long-term arsenic exposure.

To examine the association between long-term exposure to inorganic arsenic and the prevalence of hypertension, we studied a total of 382 men and 516 women residing in villages where arseniasis was hyperendemic. Hypertension was defined as a systolic blood pressure of 160 mm Hg or greater, a diastolic blood pressure of 95 mm Hg or greater, or a history of hypertension treated regularly with antihypertensive drugs. The long-term arsenic exposure was calculated from the history of artesian well water consumption obtained through standardized interviews based on a structured questionnaire and the measured arsenic concentration in well water. Residents in villages where long-term arseniasis was hyperendemic had a 1.5-fold increase in age- and sex-adjusted prevalence of hypertension compared with residents in nonendemic areas. Duration of artesian well water consumption, average arsenic concentration in drinking water, and cumulative arsenic exposure were all significantly associated with hypertension prevalence. The higher the cumulative arsenic exposure, the higher the prevalence of hypertension. This dose-response relation remained significant after adjustment for age, sex, diabetes mellitus, proteinuria, body mass index, and serum triglyceride level. The results suggest that long-term arsenic exposure may induce hypertension in humans.

Risk of internal cancers from arsenic in drinking water.

The U.S. Environmental Protection Agency is under a congressional mandate to revise its current standard for arsenic in drinking water. We present a risk assessment for cancers of the bladder, liver, and lung from exposure to arsenic in water, based on data from 42 villages in an arseniasis-endemic region of Taiwan. We calculate excess lifetime risk estimates for several variations of the generalized linear model and for the multistage-Weibull model. Risk estimates are sensitive to the model choice, to whether or not a comparison population is used to define the unexposed disease mortality rates, and to whether the comparison population is all of Taiwan or just the southwestern region. Some factors that may affect risk could not be evaluated quantitatively: the ecologic nature of the data, the nutritional status of the study population, and the dietary intake of arsenic. Despite all of these sources of uncertainty, however, our analysis suggests that the current standard of 50 microg/L is associated with a substantial increased risk of cancer and is not sufficiently protective of public health.

Comparison of the effect of acetylsalicylic acid on platelet function in male and female patients with ischemic stroke.

The aim of this study was to observe whether acetylsalicylic acid (ASA) had different effects in both sexes. Out of the ischemic stroke patients who were admitted to the National Taiwan University Hospital (NTUH), those who had not taken ASA or ASA-like drugs for more than 2 weeks were selected for this study. For the diagnosis of ischemic stroke, computed tomography (CT) of the brain was performed in all cases, and for differential diagnosis, other necessary procedures were employed in a few cases. The serum salicylate (SA) level was measured by Trinder's method, thromboxane B2 (TXB2) and 6-keto-PGF1 alpha by radioimmunoassay, threshold concentration of adenosine diphosphate (ADP) by Born's method, and circulating platelet aggregates (CPA) by Wu and Hoak's method. The present study showed that the means of serum SA levels after administration of the same dose of ASA were not significantly different between the two sexes. After ingestion of ASA, a single dose of 75 mg, 300 mg or 600 mg, or 300 mg 4 times a day, mean plasma TXB2 levels were significantly suppressed and mean threshold concentrations of ADP were significantly elevated in the two sexes. After administration of above-mentioned various doses of ASA, the abnormally high plasma TXB2 levels and abnormally low threshold concentrations of ADP and CPA ratios were significantly normalized in both male and female patients. Plasma 6-keto-PGF1 alpha levels were not influenced by ingestion of ASA 75 mg, but significantly depressed by administration of ASA 300 mg in both sexes. There were no sex differences in the antiplatelet effect of ASA in this experiment.

Effect of ethanol on the disposition of paraquat.

Rabbits that received paraquat (200 mg/kg) and ethanol (1.0-3.8 g/kg) by gavage resulted in shorter median survival times and higher mortality rates than those dosed with paraquat alone. Plasma paraquat levels in rabbits dosed with ethanol before, after and concurrently with ingestion of paraquat were significantly higher when compared with animals without ethanol intake. Furthermore, substantial differences in paraquat kinetics, higher AUC, lower total clearance (CL/F) and volume of distribution (V/F) were seen in the ethanol-paraquat group. The effect of ethanol on acute paraquat toxicity is, at least in part, associated with the increase in paraquat absorption and/or the decrease in paraquat excretion.

Determination of paraquat in tissue using ion-pair chromatography in conjunction with spectrophotometry.

Homogenized tissue was deproteinized with sulfuric acid. Paraquat in the supernatant was quantificated directly with the dithionite reagent (step 1) or concentrated by the XAD-2 column chromatographic technique before paraquat determination (step 2). Tissue paraquat levels in the range of 0.01-75 mg/kg could be quantificated by second-derivative or zero-order spectroscopy using 2.5 g of tissues. The sensitivity could be increased tenfold by using 25 g of tissue samples. The coefficients of variation of within-run and day-to-day precisions of spiked paraquat in tissue homogenates were below 5% at concentrations of 10.0, 1.0 and 0.1 mg/kg, respectively. The recoveries of the spiked paraquat in tissues ranging from 0.1-10 mg/kg were 91% by step 1 and 74% by step 2. Using these simple methods, steps 1 and 2, the paraquat concentrations in the psoas muscle, liver, lung and kidneys of a swine dosed with 0.16 g/kg of paraquat were investigated. The results were in close agreement with those of the TCA deproteinization method followed by cation-resin column chromatography. The proposed method offers the advantages of simplicity, rapidity, reasonable sensitivity and a wide range of concentrations.

Determination of paraquat from formalin-fixed tissue.

In this paper, a sulfuric acid digestion method and a clean-up technique by using cation exchange resin followed by XAD-2 resin has been developed for the determination of paraquat from formalin-fixed tissue at the submicrograms per gram level. Formalin-fixed tissue is dissolved by hot sulfuric acid, then paraquat is isolated and purified with cation exchange chromatography. The eluted paraquat forms an ion-pair with sodium dodecyl sulfate, it is then adsorbed on XAD-2 resin. Paraquat is eluted, extracted and reduced with solvent mixtures, NaCl solution and dithionite reagent, respectively. The calibration graphs of zero-order and second-derivative spectroscopy are linear in the range of 0.01-5.0 mg/kg. The relative standard deviation was less than 5% and the detection limit was 0.02 mg/kg based on 0.5-g samples. The sensitivity of the proposed method could be increased by using larger sample sizes. The method was precise and gave a quantitative recovery of paraquat spiked into formalin-fixed liver homogenates (78%). The proposed method has been satisfactorily applied to the determination of paraquat in the formalin-fixed tissues of suspected poisoned cases. It has been shown to be of great value in the field of forensic toxicology especially when formalin-fixed tissue only is available.

Spectra interference between diquat and paraquat by second derivative spectrophotometry.

A rapid and accurate method, combining solid-phase extraction and second-order derivative spectrophotomety approaches, is developed for the simultaneous determination of diquat (DQ) and paraquat (PQ) in blood, tissue and urine samples. Supernatant resulting from the precipitation of protein (with trichloroacetic acid) in plasma and tissue or Amberlite IRA-401 resin treated urine are passed through a mini-column packed with Wakogel gel (Silica gel). Analytes are then eluted with a non-organic solvent, 0.2mol/l HCl solution containing 2mol/l NH(4)Cl. UV spectrum of the eluent in 220-350nm range provides effective screen to detect the presence of DQ and/or PQ. In the presence of DQ or PQ alone, the analyte present is quantitated by conventional zero- or second-order derivative spectrophotometry. The calibration curve in the 0.1-5.0mg/l range for either analyte obeys Beer's law. When both DQ and PQ are present, their concentrations are determined by the peak amplitudes of their respective second-derivative spectra after the addition of alkaline dithionite reagent. Interference is negligible when the DQ/PQ concentration ratio is within the 5.0-0.2 range. Using a 2-ml of sample size, the detection limits for DQ and PQ in plasma are 0.02 and 0.005mg/l. The corresponding detection limits for urine samples (10ml sample size) are 0.004 and 0.001mg/l. Recoveries of DQ and PQ in triplicate plasma and urine samples spiked with 0.5mg/l of analytes are 93 and 85%. The precision of the proposed method resulting from triplicate study of spiked urine samples varies from 3.2 to 4.6% at 0.5mg/l of DQ and PQ, respectively.

Pitfalls when determining tissue distributions of organophosphorus chemicals: sodium fluoride accelerates chemical degradation.

This paper describes the tissue distributions of dichlorvos, an organophosphate, and chlorpyrifos-methyl, an organophosphorothioate, in a male individual who died after ingesting an insecticidal preparation containing these chemicals and the results of an in vitro stability study on dichlorvos and chlorpyrifos-methyl in blood and buffers. Tiny amounts of dichlorvos, 0.067 and 0.027 mg/L, were detected in the vitreous humor and cerebrospinal fluid, respectively. Although dichlorvos (0.082-8.99 mg/L or mg/kg) was detected in the thoracic aortic blood, thoracic inferior vena caval blood, pericardial fluid, bile, and spleen, it was strongly suggested that it had diffused postmortem from the stomach, which contained 879 mg, because no dichlorvos was detected in the other blood samples and tissues tested. Substantial amounts (0.615-4.15 mg/L) of chlorpyrifos-methyl were detected in all blood samples, and the order of its concentrations was as follows: pulmonary vessel blood > thoracic inferior vena caval blood > blood in the right cardiac chambers > blood in the left cardiac chambers approximately thoracic aortic blood > right femoral venous blood. The total amount of chlorpyrifos-methyl in the stomach was 612 mg. However, it was strongly suggested that virtually no chlorpyrifos-methyl diffused from the stomach into surrounding fluids and tissues postmortem because no chlorpyrifos-methyl was detected in the bile and little was found in the pericardial fluids. Neither compound was detected in the urine. In vitro experiments showed that dichlorvos (10 mg/L) almost disappeared from fresh (pH 7.4) and acidified (pH 6.2) blood samples within 24 and 72 h, respectively. However, 53 and 77% of the original amount of dichlorvos in 0.05M phosphate buffers at pH 7.4 and 6.2 were detected 72 h later. Chlorpyrifos-methyl (1 mg/L) was very stable in blood samples, regardless of the pH, during the 72-h study period, but in the pH 7.4 and 6.2 phosphate buffers, approximately 80% of the original amount had degraded after 72 h. These results indicate that organophosphates are degraded more rapidly by esterase activities than by chemical mechanisms and that organophosphorothioates are hydrolyzed chemically in aqueous solutions but are very stable in biological specimens and not metabolized by esterases. When sodium fluoride was added to blood samples, dichlorvos degraded completely within 15 min, and chlorpyrifos-methyl became very unstable. Thus, when analyzing samples to detect organophosphorus chemicals, this common preservative should not be added to fluid specimens.

Chemical derivatization and the selection of deuterated internal standard for quantitative determination--methamphetamine example.

Use of an isotopic analogue of the analyte as the internal standard in a quantitative gas chromatography-mass spectrometry targeted-compound-analysis protocol is often hindered by the availability of an adequate number (typically three for the drug/metabolite and two for the isotopic analogue) of sufficiently high mass ions that can be attributed to each member of the pair and are sufficiently free of interference by the contribution from the other component of the pair, a phenomenon termed "cross-contribution". Methamphetamine (MA) is selected as the exemplar compound to examine the effectiveness in using different chemical derivatization routes to produce derivatized analyte-isotopic analogue pairs that can generate more favorable mass spectrometric data to meet this analytical requirement. Trimethylsilyl-, trichloroacetyl-, and pentafluoropropionyl-derivatization and MA-d5, MA-d8, and MA-d9 are studied. Data resulting from this study indicate that the number of ion pairs suitable for quantitation and the degree of cross-contribution of these ions vary significantly. These data empirically demonstrate that derivatization methods play a significant role in deciding which deuterated analogue of the analyte provides the most suitable ion pairs that cause the least cross-contribution. The most suitable internal standard varies with the derivatization route adapted for an analytical protocol.

Effects of ethanol on pharmacokinetics and intestinal absorption of paraquat in animals.

The effect of ethanol on pharmacokinetics of paraquat was studied in rabbits by intravenous (i.v.) bolus injection of ethanol 0.5 g/kg (or normal saline, in the control group) followed by an i.v. dose of paraquat 20 mg/kg. A greater apparent volume of distribution (Vd), faster distribution rate constants and lower peak plasma concentration (P < 0.01) of paraquat were observed in the ethanol-treated than that in the control rabbits. The total clearance of paraquat was not significantly different between the two groups. The effect of ethanol on the intestinal absorption of paraquat was estimated by in situ intestinal perfusion technique in rats. The perfusate contained paraquat 50 micrograms/ml alone or with 1% or 2% ethanol. Inulin 320 micrograms/ml was added to the perfusate for the measurement of water net flux. The absorption clearance of paraquat as well as the absorption of water increased (P < 0.01) about two-fold in the presence of ethanol. The results of this study suggest that ethanol may potentiate paraquat toxicity by increasing intestinal absorption and tissue distribution. The critical lethal plasma concentration of paraquat is supposed to be lower in the presence of ethanol owing to increased volume of distribution.

Determination of paraquat in biologic materials by a simplified solid phase extraction and spectrophotometry.

A rapid measurement of low paraquat levels in body fluids is important for diagnosis and prognosis of paraquat intoxication. A simple and sensitive method for the determination of submicrogram levels of paraquat by solid phase extraction in conjunction with spectrophotometry has been developed. Serum, urine and formalin from fixed tissue were passed through this column and paraquat was eluted with 2 mL 2N NaOH. The eluent was reduced with dithionite and spectrophotometrically quantified with the zero- or second-order derivative. The detection limits of paraquat by this method are 0.05 mg/L and 0.01 mg/L with the zero-order and 0.005 mg/L and 0.001 mg/L with the second-order derivative, using 2 mL of serum and 10 mL of urine or formalin. The proposed method prevents the interference of biologic specimens as well as providing high extraction efficiency and good precision. The entire procedure can be completed within 30 minutes without special skills, reagents or equipment. These results show that this method is suitable for clinical and forensic toxicologic purposes.

Serum copper and zinc levels in patients with cervical cancer.

The serum copper (SCL) and zinc (SZL) levels were measured in 99 patients with cervical cancer and 50 patients with uterine myoma as controls. The mean SCL in the control group was 109.4 +/- 17.4 micrograms/ml as compared to 117.1 +/- 14.6 micrograms/dl and was not significant (NS) in 17 carcinoma in situ (CIS) patients, 142.3 +/- 14.2 micrograms/dl in 30 stage I patients (p less than 0.001), 159.0 +/- 16.6 micrograms/dl in 22 stage II patients (p less than 0.001), 171.6 +/- 25.7 micrograms/dl in 10 stage III or IV patients (p less than 0.001), and 166.2 +/- 32.2 micrograms/dl in 20 recurrent patients (p less than 0.001). The SCL returned to control level 2 weeks after surgical treatment for the stage I and II patients (mean 110.6 +/- 19.6 and 108.7 +/- 20.4 micrograms/dl, respectively, p less than 0.001). The SZL was 97.2 +/- 15.8 micrograms/dl in control patients and only showed a significant decrease in stage III or IV and recurrent patients (67.2 +/- 16.6 and 70.4 +/- 17.2 micrograms/dl, respectively). Concerning the copper/zinc ratio, the control group was 1.13 +/- 0.07 as compared to 1.17 +/- 0.07 in CIS (p = 0.06), 1.51 +/- 0.24 in stage I (p less than 0.001), 1.85 +/- 0.37 in stage II (p less than 0.001), 2.66 +/- 0.61 in stage III or IV (p less than 0.001), and 2.50 +/- 0.75 in recurrent patients (p less than 0.001). Taking mean +/- 2.5 SD of the control values as cut off points, the percentages of the recurrent patients with abnormal SCL, SZL, and a Cu/Zn ratio were 65, 30 and 90%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of tissue paraquat into formalin solution during fixation.

Formalin-fixed tissues and formalin solutions are among the most frequently found materials in pathology and forensic science laboratories. However, these materials are seldom used for the identification of poisons for forensic toxicology purposes. In this study, the possibility that paraquat may be released from formalin-fixed tissues during the fixation process was investigated. However, because of the interference of formaldehyde on the reduction of paraquat with dithionite reagent, paraquat in formalin solutions was treated with ion-pair column chromatography and then determined by measuring the derivative spectrum of reduced paraquat. The results show that the interference of formalin on paraquat determination has been eliminated by the proposed method. Furthermore, a study on the formalin solutions of fixed organs in cases with suspected paraquat intoxication revealed that portions of tissue paraquat had been released into formalin during the fixation process. Moreover, the paraquat levels in formalin increased with increased storage time. Therefore, these data suggest that the combined concentrations of paraquat in the formalin-fixed tissues and formalin solutions might reflect more reliably the total paraquat in the postmortem tissues. This investigation could be of value to the forensic toxicologist, especially in cases in which no fresh tissue samples are available for analysis.

Effect of ethanol on acute paraquat toxicity in rabbits.

The effects of simultaneous administration of ethanol and paraquat on the blood paraquat levels and fatality in rabbits were investigated to evaluate the role of alcohol ingestion in inducing susceptibility to paraquat intoxication. Paraquat alone and with the combination of ethanol were administered orally to rabbits. Rabbit mortality rates for the 24 h period following the ingestion of paraquat alone and paraquat combined with 2.0 and 3.8 g/kg of ethanol, were 0/8, 4/8 and 8/8 subjects, respectively. Animals which ingested paraquat and ethanol at 2.0 and 3.8 g/kg showed significant elevation of blood paraquat levels. Furthermore, an animal dosed with ethanol 2 h following the ingestion of paraquat, consequently, exhibited a marked increase in paraquat levels in the blood 0.5 h after the administration of the ethanol. On the other hand, the average values of Tmax, Cmax, and AUC 8-8 h of paraquat in rabbits dosed simultaneously with paraquat (200 mg/kg) and ethanol, at 2 g/kg, were 1.38 +/- 0.52 h, 28.11 +/- 14.08 micrograms/ml, and 108.97 +/- 39.54 micrograms/ml.h, and at 3.8 g/kg of ethanol, were 2.20 +/- 1.10 h, 47.61 +/- 19.15 micrograms/ml, and 194.43 +/- 53.82 micrograms/ml.h, respectively. These values were significantly higher than those for the rabbits dosed with 200 mg/kg of paraquat alone (Tmax, Cmax, and AUC 0-8 h were 0.94 +/- 0.18 h, 13.81 +/- 2.71 micrograms/ml, and 44.1 +/- 8.6 micrograms/ml.h, respectively). These results show a close correlation between blood paraquat levels in rabbits dosed simultaneously with ethanol and mortality rates.(ABSTRACT TRUNCATED AT 250 WORDS)