Enzymes involved in the bioactivation of 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone in patas monkey lung and liver microsomes

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen in animals. Our previous studies indicated that there are differences between rodents and humans for the enzymes involved in the activation of NNK. To determine if the patas monkey is a better animal model for the activation of NNK in humans, we investigated the metabolism of NNK in patas monkey lung and liver microsomes and characterized the enzymes involved in the activation. In lung microsomes, the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK- N-oxide), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was observed, displaying apparent Km values of 10.3, 5.4, 4.9, and 902 microM, respectively. NNK metabolism in liver microsomes resulted in the formation of keto aldehyde, keto alcohol, and NNAL, displaying apparent Km values of 8.1, 8.2, and 474 microM, respectively. The low Km values for NNK oxidation in the patas monkey lung and liver microsomes are different from those in human lung and liver microsomes showing Km values of 400-653 microM, although loss of low Km forms from human tissue as a result of disease, surgery or anesthesia cannot be ruled out. Carbon monoxide (90%) significantly inhibited NNK metabolism in the patas monkey lung and liver microsomes by 38-66% and 82-91%, respectively. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) and aspirin (a cyclooxygenase inhibitor) decreased the rate of formation of keto aldehyde and keto alcohol by 10-20 % in the monkey lung microsomes. Alpha-Napthoflavone and coumarin markedly decreased the oxidation of NNK in monkey lung and liver microsomes, suggesting the involvement of P450s 1A and 2A6. An antibody against human P450 2A6 decreased the oxidation of NNK by 12-16% and 22-24% in the patas monkey lung and liver microsomes, respectively. These results are comparable to that obtained with human lung and liver microsomes. Coumarin hydroxylation was observed in the patas monkey lung and liver microsomes at a rate of 16 and 4000 pmol/min/mg protein, respectively, which was 5-fold higher than human lung and liver microsomes, respectively. Immunoblot analysis demonstrated that the P450 2A level in the individual patas monkey liver microsomal sample was 6-fold greater than in an individual human liver microsomal sample. Phenethyl isothiocyanate, an inhibitor of NNK activation in rodents and humans, decreased NNK oxidation in the monkey lung and liver microsomes displaying inhibitor concentration resulting in 50% inhibition of the activity (IC50) values of 0.28-0.8 microM and 4.2-6.8 microM, respectively. The results demonstrate the similarities and differences between species in the metabolic activation of NNK. The patas monkey microsomes appear to more closely resemble human microsomes than mouse or rat enzymes and may better reflect the activation of NNK in humans.      

Chemopreventive activity of thiol conjugates of isothiocyanates for lung tumorigenesis

A series of L-cysteine (L-Cys), glutathione (GSH), and N-acetyl-L- cysteine (NAC) conjugates of phenethyl (PEITC), benzyl (BITC), and 6- phenylhexyl isothiocyanate (PHITC) were studied for their inhibitory activity toward metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mouse lung microsomes. Selected compounds, PEITC, PEITC-GSH, PEITC-NAC and PHITC-NAC, were also assayed for the potential chemopreventive activity toward NNK-induced lung tumorigenesis in A/J mice. Results showed that PEITC and its conjugates inhibited NNK metabolism with decreasing potency: PEITC < PEITC-GSH > PEITC-Cys > PEITC-NAC. PHITC and its GSH and NAC conjugates exhibited nearly 10 times higher inhibitory activity toward NNK metabolism than the PEITC counterparts. In the tumor bioassay, as expected, the conjugates exhibited inhibitory activity against lung tumorigenesis induced by NNK. PEITC-GSH was not inhibitory at 4 micromol/mouse, but it inhibited approximately 32% of lung tumor multiplicity at 8 micromol/mouse. PEITC-NAC at 5 and 20 micromol/mouse both inhibited approximately 30% tumor multiplicity. Among all the conjugates examined, PHITC-NAC was the most potent. At a 5-micromol dose, it completely inhibited tumor multiplicity and incidence to the background level observed in the control group. These results revealed that the structure-activity relationships of the conjugates are similar to those found with their parent isothiocyanates (ITCs), i.e., the potency increased with the increasing alkyl chain length from two to six carbons in arylalkyl ITCs, suggesting that a common active species is involved. The inhibitory activity of ITC conjugates and the expected low toxicity make thiol conjugates of ITC a promising new series of chemopreventive agents.      

Detection of hepatic metastases: analysis of pulse sequence performance in MR imaging

Forty-three patients with liver metastases were imaged using 14 different pulse sequences (average, 7.5 sequences per patient) to allow direct comparison of their performance. "T2-weighted" spin-echo (SE) images, "T1-weighted" inversion recovery (IR) images, and "T1-weighted" SE images were obtained using a wide range of timing parameters. Pulse sequence performance was quantitated by measuring liver signal-to-noise (S/N) ratios and cancer-liver signal difference-to-noise (SD/N) ratios. Data were standardized to reflect a constant imaging time of 9 minutes for all pulse sequences. The SE 2,000/120 (TR [repetition time]/TE [echo time]) sequence resulted in the greatest SD/N ratio of the T2-weighted SE sequences but also yielded the low S/N ratios, poor anatomic resolution, and motion artifacts common to all T2-weighted SE images. IR sequence images were also sensitive to motion artifacts because of the use of a long TR (1,500 msec). Short TR/TE T1-weighted SE sequences (SE 260/18) had the greatest SD/N ratio (P less than .05), S/N ratio, and anatomic resolution. Furthermore, extensive signal averaging appears to be a powerful solution to all types of motion artifacts in the abdomen.     

Characterisation of renal chloride channel, CLCN5, mutations in hypercalciuric nephrolithiasis (kidney stones) disorders

Mutations of the renal-specific chloride channel (CLCN5) gene, which is located on chromosome Xp11.22, are associated with hypercalciuric nephrolithiasis (kidney stones) in the Northern European and Japanese populations. CLCN5 encodes a 746 amino acid channel (CLC-5) that has approximately 12 transmembrane domains, and heterologous expression of wild-type CLC-5 in Xenopus oocytes has yielded outwardly rectifying chloride currents that were markedly reduced or abolished by these mutations. In order to assess further the structural and functional relationships of this recently cloned chloride channel, additional CLCN5 mutations have been identified in five unrelated families with this disorder. Three of these mutations were missense (G57V, G512R and E527D), one was a nonsense (R648Stop) and one was an insertion (30:H insertion). In addition, two of the mutations (30:H insertion and E527D) were demonstrated to be de novo, and the G57V and E527D mutations were identified in families of Afro-American and Indian origin, respectively. The G57V and 30:H insertion mutations represent the first CLCN5 mutations to be identified in the N-terminus region, and the R648Stop mutation, which has been observed previously in an unrelated family, suggests that this codon may be particularly prone to mutations. Heterologous expression of the mutations resulted in a marked reduction or abolition of the chloride currents, thereby establishing their functional importance. These results help to elucidate further the structure-function relationships of this renal chloride channel.      

The role of computed tomography in the diagnosis of arterial gas embolism in fatal diving accidents in Tasmania

Four cases of fatal diving accidents in Tasmania are presented, highlighting the role of CT in the investigation of diving fatalities. The CT technique allows rapid diagnosis when arterial gas embolism (AGE) is suspected. The traditional method of investigation, underwater autopsy, is a difficult procedure that requires specialized training in which the subtle diagnosis of AGE may be completely missed. Facilities for performing underwater autopsies are normally available only in tertiary referral centres, and therefore the diagnosis of AGE may be missed due to lack of facilities. The use of CT in the diagnosis of AGE in divers was first utilized in the early 1980s but has still not become widely adopted in forensic practice. This radiological technique has the advantage of being sensitive, quick, reliable, readily available and provides a permanent record. For hospitals that do not have a resident forensic pathologist, a CT scan can be easily performed and interpreted to eliminate the possibility of AGE. There are a number of pitfalls in the diagnosis of AGE with CT, particularly intravascular gas production following postmortem fermentation and off-gassing. Awareness of these pitfalls will help the radiologist in making a correct diagnosis of AGE.     

毛细管气相色谱法测定复方利福平片中异烟肼和吡嗪酰胺的含量

目的：建立复方利福平片中异烟肼和吡嗪酰胺的含量测定方法。方法：毛细管气相色谱法,以乙酰苯胺为内标,色谱柱为弹性石英毛细管柱２５ｍ ×０ ．３２ｍ ｍ ×０ ．５２μｍ （ＤＢ１） ;柱温１７０ ℃;气化室温度２５０ ℃;检测器温度２５０ ℃;载气为高纯氮。结果：异烟肼和吡嗪酰胺的线性范围分别是０ ．４２６ ～２ ．１３ｍｇｍｌ 和１ ．２３ ～６ ．１３ｍｇｍｌ;平均回收率分别为９９ ．８１ ％ 和９９ ．６３ ％ 。结论：方法简便,快速,准确,可作为该制剂的检测方法。     

Construction of an ultrasound biopsy phantom

The technique for construction of an agar-based ultrasound biopsy phantom is described. Features include tissue equivalent reflectivity, long life and non-shadowing targets. The phantom is useful for learning the necesary co-ordination between needle and probe for ultrasound needle guidance. This skill should initially be practised in vitro, on a device such as this.     

Rate of radial bone mineral accretion in healthy children

Radial bone width and mineral content were measured in 392 healthy Cambridge children aged 6–12 years from a cohort of 420 children studied 2 years previously. The typical rate of bone mineral accretion was 0.044 g/cm/year for boys across the whole age range and 0.042 g/cm/year for girls up to a mean age of 9 years, rising rapidly thereafter in association with changes in body size. The factors best predicting bone mineral accretion rate were mean bone mineral content, mean height, height velocity, weight velocity and bone width velocity. After adjusting for anthropometry, age did not contribute to the model. These are the first available longitudinal reference data for the rate of radial bone mineral accretion in healthy children.     

Pelvic ultrasound measurements in normal girls

Pelvic ultrasound scans were carried out in 153 normal girls aged between 3 days and 14.9 years, in order to obtain reference data for ovarian volume, uterine length and uterine configuration. The right ovary was significantly larger than the left (by about 17%). Ovarian volume increased exponentially with age, over this age range. No relationship with pubertal stage (independent of age) could be demonstrated. Uterine length decreased from birth to 4 years, before steadily increasing. The fundalcervical ratio (FCR) decreased initially then increased to lie above 1.0 by 15 years of age. A midline endometrial echo was seen in half of the subjects aged less than 6 months, but otherwise it was not seen before 11.8 years of age or at Tanner stage B2. Smoothed reference centile curves for uterine length, right and left ovarian volume were produced, allowing z scores (or SD scores) to be calculated for each measurement.