Kinetics of acetyl CoA: Arylamine N‐acetyltransferase from rapid and slow acetylator fish tissues

N‐acetyltransferase (NAT) activity was determined in 100 fish (Oreochromis mossambicus) livers using 2‐aminofluorene and p‐aminobenzoic acid as substrates. Overall, the liver NAT activity of the 50 females was higher than the liver NAT activity of the 50 males. The activities (mean ± SD) of NAT from kidney, blood, intestine, and liver of males was 0.42 ± 0.11, 0.12 ± 0.03, 0.12 ± 0.08, and 1.56 ± 0.54 nmol/min/mg protein for the acetylation of 2‐aminofluorene and 0.36 ± 0.09, 0.09 ± 0.01, 0.11 ± 0.04, and 0.46 ± 0.15 nmol/min/mg protein for the acetylation of p‐aminobenzoic acid. In kidney, blood, intestine, and liver from female fish, the activities obtained were 1.60 ± 0.12, 0.35 ± 0.07, 0.15 ± 0.09, and 1.89 ± 0.50 nmol/min/mg protein for 2‐aminofluorene and 0.95 ± 0.11, 0.27 ± 0.03, 0.13 ± 0.09, and 0.57 ± 0.12nmol/min/mg protein for p‐aminobenzoic acid. Kinetic constants for arylamine N‐acetyltransferase activity in kidney, blood, intestine, and liver from fish with rapid, intermediate, and slow acetylator activity were determined. Apparent K _m and V _max values for 2‐aminofluorene were 5.5 and 7‐fold higher for liver than for the other tissues. Apparent K _m and V _max values for p‐aminobenzoic acid were 3.5 and 4.7‐fold higher for liver than for the other tissues. Based on the 2‐aminofluorene NAT activity of liver, there appears to be a polymorphism in NAT activity with 16 rapid, 28 intermediate, and 56 slow acetylators among the 100 fish assayed. This is the first demonstration of acetyl CoA: arylamine N‐acetyltransferase activity in fresh water fish and could lead to the development of a fish model for monitoring the effect of pollution of water environments on native species.     

Paeonol promotes arylamine N‐acetyltransferase activity in human bladder tumor cells

Paeonol was used to determine effects of arylamine N‐acetyltransferase (NAT) activity in human bladder tumor cells. The NAT activity was measured by high performance liquid chromatography assaying for the amounts of N‐acetyl‐2‐aminofluorene (2‐AAF) and N‐acetyl‐p‐amino‐benzoic acid (N‐Ac‐PABA) and remaining 2‐aminofluorene (2‐AF) and p‐aminobenzoic acid (PABA). The NAT activity in human bladder tumor cells was promoted by paeonol in a dose‐dependent manner, i.e. the higher the concentration of paeonol, the higher the promotion of NAT activity. The data also indicated that paeonol increases the apparent values of K_m and K_max from human bladder tumor cells in cytosols and intact cells. This report is the first demonstration to show paeonol did promote human bladder tumor cell NAT activity.     

Determination of phenol,cresols and xylenols in workplace air using XAD‐2 sorbent cartridges

A method for sampling and analysis of phenolic compounds in workplace atmospheres using Amberlite XAD‐2 sorbent tubes has been laboratory and field tested. After extraction of the adsorbed phenols with methylene chloride, the concentrated extract was analyzed by capillary column gas chromatography coupled with a flame ionization detector or a mass spectrometer. For a 3L air sample, the method has a detection limit for phenol, cresols, alkyl‐substituted phenols and xylenols of 0.3 mg/m³ using gas chromatography with flame ionization detection (GC/FID) analysis and 0.07 mg/m³ using gas chromatography mass spectroscopy/selected ion monitoring GC‐MS/SIM. Evaluation tests conducted in the laboratory indicated recoveries >85% for a wide range of phenols, cresols, xylenols and other alkyl‐substituted phenols and resorcinol. Sampling conditions were established to ensure that no breakthrough occurred for a predetermined sample volume of three (3) liters even in humid atmospheres. Stability tests indicated that for storage of up to 2 weeks at 4°C, only resorcinol showed a significant reduction in sample recovery. The proposed method and NIOSH Method 3502 were compared for the determination of phenol concentrations in air samples collected at a plant processing phenolic resins. Phenol was positively identified in the XAD sample using GC‐MS/SIM and was detected at concentrations of 40–50% of the total phenols detected using NIOSH Method 3502. This difference is attributed to the airborne paniculate matter which is known to contain bisphenols that will contribute to the total phenolic content sampled in Method 3502, but not on the XAD sample.     

Light‐induced changes in rabbit pineal gland n‐acetyltransferase activity

N‐acetyltransferase (NAT) activity was determined in different ages of New Zealand White rabbit pineal gland using 2‐aminofluorene and p‐aminobenzoic acid as substrates and it was assayed by high pressure liquid chromatography. Rabbits of different ages were either sacrificed during the light phase, exposed to darkness or light for 1 min during the dark phase of the lighting cycle, returned to their cages in darkness for 30 min and then sacrificed. Pineal gland NAT activity in animal nocturnally exposed to 1 min of light was inhibited in animals 1 ‐day‐old of age. Nocturnal light exposure did not inhibit enzyme activity in 1‐day‐old rabbit, even though these animal displayed clear light : dark differences in pineal gland NAT activity. Nocturnal light exposure also did not inhibit night time levels of NAT activity in 1‐day‐old animals who had been bilaterally enucleated. The result suggested that this effect is retinally mediated. Pre‐treatment of 1‐day‐old and 60‐day‐old animals with the isoproterenol (beta‐noradrenoreceptor agonist drug), prevented the nocturnal light‐induced inhibition of NAT activity. The different sensitivity of 60‐day‐old and 1‐day‐old animals to different illuminances or durations of nocturnal light exposure, was that the duration or intensity of light exposure was enable to inhibit nocturnal NAT activity. The NAT activity was at least 3.2‐ to 4.6‐fold greater in 1‐day‐old rabbits compared to 60‐day‐old rabbits. Kinetic constants for arylamine NAT activity in pineal gland from rabbits were determined. K_m and F_max values for 2‐aminofluorene were 2.6‐fold higher for light exposure than for no light exposure rabbits. This is the first demonstration of the retina‐pineal gland pathway appears light‐induced changes in pineal glands of animals in 1‐day‐old of ages or older; but this pathway does not function in 60‐day‐old rabbits like manner in 1‐day‐old rabbits.     

Effects of ellagic acid on arylamine N‐acetyltransferase activity in human colon tumor cells

The inhibition of arylamine N‐acetyltransferase (NAT) activity by ellagic acid was determined in a human colon tumor (adenocarcinoma) cell line. Two assay systems were performed, one with cellular cytosols (9000g supernatant), the other with intact colon tumor cell suspensions. The NAT activity in a human colon tumor cell line was inhibited by ellagic acid in a dose‐dependent manner in both types of examined systems: i.e. the greater the concentration of ellagic acid in the reaction, the greater the inhibition of NAT activities in both systems. The data also indicated that ellagic acid decreased the apparent K _m and V _max of NAT enzymes from human colon tumor cells in both the systems examined. This report is the first demonstration which showed ellagic acid affect human colon tumor cell NAT activity.     

Characterisation of cobalt‐binding proteins in occupational cobalt exposuret†

In animal experimentations cobalt is found to be carcinogenic under conditions which are comparable with a possible exposure of human beings at the workplace.^1,2 Little is known about binding and distribution of incorporated cobalt in blood.

Blood, serum and plasma of workers occupationally exposed to cobalt were analyzed concerning cobalt‐binding. The samples were focused by preparative IEF in layers of granulated gels. The cobalt concentrations in the isolated fractions were measured with the aid of flameless atomic absorption spectrometry. The whole blood samples exhibit peaks with different sizes in the pH‐5‐ and ‐7‐range. The proportion of these peaks can change with individual samples. In serum and plasma cobalt is found only in the pH‐5‐range.

When separated by gelelectrophoresis under denaturating conditions the cobalt‐fractions in all blood, serum and plasma samples shows a similar protein pattern. Several proteins with different molecular weights are detected in the pH‐5‐fractions, whereas only hemoglobin is found in the pH‐7‐fractions. In agreement with these results is the refocusing in ultrathin‐layer of polyacrylamide. Heterogeneous protein patterns are demonstrated with the pH‐5‐fractions; the pH‐7‐fractions yield only the hemoglobin pattern.

In vivo cobalt is bound to plasma proteins‐perhaps‐albumin^3,4‐and hemoglobin. Till now the chemical structure of the binding is unknown.     

Determination of pentachlorophenol in human urine using solid phase extraction and GC‐MS analysis with stable isotope dilution

A method for the determination of pentachlorophenol (PCP) in human urine was developed. Urine samples were fortified with ¹³C₆‐IabeIed PCP and hydrolyzed with sulfuric acid prior to extraction. The urine samples were then passed through preconditioned solid phase extraction cartridges containing octadecylsilane adsorbent. The PCP was subsequently eluted from the cartridge with ethyl acetate and converted to pentachloroanisole prior to GC‐MS analysis. GC‐MS quantitation was performed using the technique of stable isotope dilution. Percent recoveries averaging 100, 97, and 111% were obtained for replicate urine samples fortified with approximately 2, 20, and 200 ng/mL of PCP, respectively. The method detection limit was estimated to be 0.2 ng/mL.     

Phytoconstituents evaluation and antihyperglycemic and antihyperlipidemic effects of Mahonia leschenaultia Takeda in streptozotocin-induced diabetic rats

Hyperlipidemia is an associated complication of diabetes mellitus. Many spices and herbs are known to be hypoglycemic. Mahonia leschenaultia Takeda (Berberidaceae) (MLT) is a traditional medicinal plant that is commonly used against diseases in India and other Asian countries. The aim of this study is to identify the phytoconstituents by gas chromatography–mass spectrography (GC–MS) and evaluate the antihyperglycemic and antihyperlipidemic effects of the ethanol extract of MLT on streptozotocin (STZ)-induced diabetic rats. Phytoconstituents like oleicacid, 9,12-octadecadienoic acid (Z, Z)-n-hexadecanoic acid were identified as high % peak when compared with other compounds by GC–MS. STZ-induced diabetic animals were fed with plant extracts at different doses (250 or 500 mg kg^?1 body wt) of ethanol extract of MLT. The results of the study revealed a significant increase in total hemoglobin and glycosylated hemoglobin along with the reduction in hyperglycemia and hyperlipidemia effects in MLT-treated STZ-induced diabetic rats compared to diabetic only rats. Further, ethanol extracts of MLT-treated animals at 500 mg kg^?1 showed that shrinkage of β cells of islets of Langerhans was restored as evidenced by histological studies of pancreas of diabetic animals. Thus the ethanol extract of MLT might serve as a reliable adjuvant for antihyperglycemic and antihyperlipidemic effects of STZ, and may be promising for development of phytomedicines for diabetes mellitus.     

Chemosensory and behavioural responses of the turnip sawfly, Athalia rosae, to glucosinolates and isothiocyanates

Summary. The turnip sawfly Athalia rosae sequesters glucosinolates from its cruciferous host plants in the larval stage. Investigation of the chemosensory and behavioural responses of adult A. rosae to glucosinolates and their volatile hydrolysis products, isothiocyanates, revealed that females detect glucosinolates by contact chemoreception and isothiocyanates by antennal olfaction. In electroantennogram recordings, four isothiocyanates (allyl [2-propenyl] isothiocyanate, benzyl isothiocyanate, butyl isothiocyanate and iberverin [3-methylthiopropyl isothiocyanate]) were active at all doses presented, including the lowest (0.1 μg), whilst the threshold for detection of three others, iberin [3-methylsulphinylpropyl isothiocyanate], methyl isothiocyanate, and sulforaphane [4-methylsulphinylbutyl isothiocyanate], was higher, at between 1 and 10 μg (source concentration of volatiles). Allyl isothiocyanate attracted experienced females in a four-chambered olfactometer, whilst na?ve females showed no response. Allyl isothiocyanate also attracted mature females to baited yellow water traps in field trials, although immature females were repelled at high isothiocyanate concentrations. In laboratory behavioural bioassays the glucosinolates sinigrin (allyl [2-propenyl] glucosinolate) and sinalbin (p-hydroxybenzyl glucosinolate), stimulated ovipositor probing in mature female A. rosae to an extent comparable to hot-water extracts of their host plants. These responses show that glucosinolates and isothiocyanates play an important role in host finding and host recognition in A. rosae.     

Abstracts to forthcoming papers

Soil and sediments collected at a former chlor‐alkali plant in coastal Georgia (United States), revealed the presence of PCDF concentrations as great as 82.3 ng/g, dry wt. PCDF congener profile in soil was typical of “chlorine pattern”; with elevated proportions of OCDF and HpCDF. Concentrations of PCDFs declined gradually by 44‐fold at a distance of about 500 m along the contamination gradient. Of PCDDs/DFs, 2,3,7,8‐substituted PCDFs accounted for 94–98% of the TCDD‐like activity, which declined by 25‐fold, corresponding with the reduction of total PCDF concentrations. Concentrations of PCDDs in sediments were as great as 17 ng/g, with an elevated contribution from OCDD. The magnitude of decline in PCDD concentrations with distance from the source was less pronounced than that for PCDFs. PBDDs and PBDFs were not detected. Polybrominated biphenyl ether (PBBE) and monobromo‐heptachloro dibenzo‐p‐dioxins and dibenzofurans (PXDDs/DFs) were found, though, at low concentrations. Their spatial distribution was similar to those of PCDDs.     

Cleanup by solid‐phase extraction and HPLC of organochlorine insecticides and PCBS in various nonfatty and fatty samples

An accurate, simple and cheap extraction and cleanup procedure for capillary GC analysis of organo‐chlorine insecticides (OCs) in vegetables (cabbage and carrots) at the ng/g level, and for soil at the μg/g level is presented. The cleanup is carried out on solid‐phase extraction (SPE) cartridges, filled with 500 mg silica, 1 g of deactivated Florisil (10% w/w water), and 100 mg of anhydrous sodium sulphate. Recoveries >90% are obtained. The cleanup of OCs in fatty samples on an HPLC LiChrosorb Si 100 column is evaluated for subsequent capillary GC analysis. Fractionation of OCs and Aroclor 1254 and 1260 on an HPLC Nucleosil 100 column appears to be satisfactory.     

Removal of Diazo dye from aqueous phase by algae Spirogyra species

This communication presents a preliminary study conducted to investigate dye (Direct Brown 2‐Diazo) colour removal using viable algae Spirogyra species. The results indicate the ability of algae Spirogyra species to remove dye colour and found to be dependent on the contact time and biomass. Colour removal mechanism by algae Spirogyra species may be attributed to biosorption and/or bioconversion and/or biocoagulation.     

Environmental degradation of atrazine,linuron and fenitrothion in soil samples

The persistence of atrazine, linuron and fenitrothion in soil samples from an estuarine area (Ebro delta, Tarragona, Spain) has been studied. Soil samples from the top surface (10 cm) were collected during 1989–91, freeze‐dried, sieved through 200 μm, Soxhlet extracted with methanol, cleaned‐up with Florisil and analysed either by gas chromatography‐nitrogen phosphorus detection (GC‐NPD), in the case of atrazine and fenitrothion, or by liquid chromatography with diode array detection (LC‐DAD), for linuron. Confirmation of the samples analysed by GC‐NPD was carried out using GC‐mass spectrometric detection (MS) in the electron impact mode. The soil half lives obtained under the real environmental conditions have been calculated and the values obtained have been correlated with the physicochemical properties of each pesticide and the soil type. Degradation was affected by volatilization since temperatures in the area of study are relatively high, ca. 30°C, in the summer period. In the case of atrazine it has been shown that deethylatrazine is formed in all the samples studied..     

Occupational exposure to airborne aromatic amines in rubber manufacturing: Determination by gas chromatography‐mass spectrometry

Aromatic amines (AAs) are used in a variety of chemical industries and consequently they are the object of great attention in occupational hygiene owing to the carcinogenic effects that many of them have shown. This work outlines a procedure for the determination of occupational exposure to airborne AAs in the rubber industry and the application of this method in a tyre manufacturing plant using p‐phenylenediamines as antiozonants. Samples were collected on a glass fibre filter followed by a silica gel tube and analyzed by GC‐MS/SIM using a capillary column coated with methyl silicone. p‐Phenylendiamines and other AAs probably formed as thermodegradation products were found in concentrations of up to 10 μg/m³ during rubber vulcanization.     

Potentiometric studies of mixed complexes of cobalt(II) and copper(II) with l‐Asparagine and adenine

A potentiometric titration technique has been used to determine the stability constants for the various complexes of Co(II) and Cu(II) with L‐asparagine and from DNA base, e.g. adenine. Stability constants of ternary systems have been evaluated by the method suggested by Irwing‐Rossotti. In addition, the conditional constants were calculated as a function of pH. The maximum values of the conditional formation constants were found to be in accordance with the mixed‐ligand complex formation constants in a determined pH region. Furthermore, the molar fractions of different species from mixed complexes were calculated by means of formation constants. The values of stability constants of mixed‐ligand complexes at 25°C are as follows: log K= 5.25 for Co(II)‐L‐asparagine‐adenine; log K= 9.30 for Cu(II)‐L‐asparagine‐adenine. The ionic strength was kept constant at I = 0.20 with NaClO₄.     

Organochlorine insecticide residues in Egyptian mothers’ milk

Two hundred ninety‐two mother's milk samples were collected during 1994 from ten Egyptian governorates representing rural, metropolitan, and newly reclaimed desert areas. The samples were analyzed for 14 organochlorine insecticides and metabolites using electron capture gas chromatography and confirmed by gas chromatography/mass spectrometry (GC/MS). The overall samples average showed that the most abundant organochlorine insecticide residues were p,p'‐DDE, α‐endosulfan, β‐HCH and, p,p’‐DDT. Residues in the range of not detectable to less than 14μg/l whole milk were recorded for α‐HCH, heptachlor, heptachlor epoxide, aldrin, dieldrin, endrin endrin aldehyde and p,p'‐DDD. The percentage of samples exceeding the acceptable daily intake for children (ADIs) set by the FAO/WHO ranged from 19% to 44% in all governorates included in the study except the governorate of Minia in which only 2.6% of the samples exceeded the ADI values. The insecticides exceeding ADI values are mostly heptachlor and heptaclor epoxide (ADI = 3.12 μg/1 whole milk).     

Effects of the Ellagic acid on the N‐Acetyltransferase activity and Acetylation of 2‐aminofluorene in the rat

The effects of the ellagic acid on the in vitro and in vivo acetylation of 2‐aminofluorene were investigated in male Sprague‐Dawley rats. For in vitro examination, cytosols with or without ellagic acid co‐treatment showed different percentage of 2‐aminofluorene acetylation. For in vivo examination, pre‐treatment of male rats with ellagic acid (10 mg/kg) 24h prior to the administration of 2‐aminofluorene (50 mg/kg) resulted in a 26% and 29%, respectively, decrease in the urinary and fecal recovery of N‐acetyl‐2‐aminofluorene, and a 37% decrease in the metabolic clearance of 2‐aminofluorene to N‐acetyl‐2‐aminofluorene. This is the first demonstration that ellagic acid decrease the N‐acetylation of carcinogens in vitro and in vivo.     

Instrument tuning for trace analysis of dioxins and furans for the vg 70s/se gas chromatography/mass spectrometry system using electron impact mode

We present the salient characteristics of tuning procedures that have evolved from our 3 years of experience while producing large amounts of data of the highest quality. Our laboratory routinely analyzes 2,3,7,8‐substituted dioxins and furans at parts per quadrillion levels in 50–100 g of human serum samples using the VG 70S/SE stand‐alone gas chromatography/mass spectrometry (GC/MS) system at 10000 resolution (defined by 5% overlap) in the selected ion recording mode. Trace measurement of dioxins and furans in human biological matrices—for example, whole blood, serum, or adipose tissue‐requires reproducible instrument tuning within day as well as among days for quality results. Additionally, instument‐tuning procedures among operators must be reproducible to minimize operator bias because periodically different operators are used. Instrument tuning is highly subjective and argumentive; therefore, to minimize the ambiguity within our laboratory, we have standardized our technique to reproducibly tune the GC/MS system.     

Optimal Allocation of Conservation Resources to Species That May be Extinct

Abstract: Statements of extinction will always be uncertain because of imperfect detection of species in the wild. Two errors can be made when declaring a species extinct. Extinction can be declared prematurely, with a resulting loss of protection and management intervention. Alternatively, limited conservation resources can be wasted attempting to protect a species that no longer exists. Rather than setting an arbitrary level of certainty at which to declare extinction, we argue that the decision must trade off the expected costs of both errors. Optimal decisions depend on the cost of continued intervention, the probability the species is extant, and the estimated value of management (the benefit of management times the value of the species). We illustrated our approach with three examples: the Dodo (Raphus cucullatus), the Ivory‐billed Woodpecker (U.S. subspecies Campephilus principalis principalis), and the mountain pygmy‐possum (Burramys parvus). The dodo was extremely unlikely to be extant, so managing and monitoring for it today would not be cost‐effective unless the value of management was extremely high. The probability the Ivory‐billed woodpecker is extant depended on whether recent controversial sightings were accepted. Without the recent controversial sightings, it was optimal to declare extinction of the species in 1965 at the latest. Accepting the recent controversial sightings, it was optimal to continue monitoring and managing until 2032 at the latest. The mountain pygmy‐possum is currently extant, with a rapidly declining sighting rate. It was optimal to conduct as many as 66 surveys without sighting before declaring the species extinct. The probability of persistence remained high even after many surveys without sighting because it was difficult to determine whether the species was extinct or undetected. If the value of management is high enough, continued intervention can be cost‐effective even if the species is likely to be extinct.     

Metalaxyl—its persistence and metabolism in soil

Different biotic and abiotic factors were found to play a vital role in attenuating metalaxyl residues in soil. In addition to metalaxyl many other products were found by HPLC and GC‐MS analysis while studying its soil metabolism in presence of natural sunlight. Three compounds were identified and characterized: 2,6‐dimethylaniline, 2,6‐dimethyl‐N‐ethylacetanilide and N‐(2,6‐dimethyl phenyl) alanine methyl ester.