Structure and vibrational spectra of mononitrated benzo[a]pyrenes

The molecules benzo[a]pyrene (BaP) and 1-, 3-, and 6-nitrobenzo[a]pyrene (1-NBaP, 3-NBaP, 6-NBaP) are currently of significant interest due to their presence in respirable combustion exhaust particulates and their mutagenic and carcinogenic properties. Structure-function correlations as well as spectroscopic signatures for trace analysis are necessary for these benzo[a]pyrene derivatives. In this paper, detailed infrared and Raman spectroscopic data of BaP and its three mononitrated isomers are provided for the first time. By utilizing density functional theory (DFT, B3LYP method with 6-311+G basis set), the molecular geometries and the vibrational spectra are calculated. Good agreement is noted between the calculated and experimental geometry for BaP, and predictions of the vibrational data for all compounds are within approximately 5 cm-1 of the experimental data. Normal mode assignments are proposed with particular emphasis on the nitro group vibrations. The geometrical distortions of the BaP structure upon nitro group substitution and correlations between structural parameters and vibrational data as well as structure-function relationships related to the mutagenicity of this important class of polycyclic aromatic hydrocarbons are discussed.     

Identification and quantification of urinary benzo[a]pyrene and its metabolites from asphalt fume exposed mice by microflow LC coupled to hybrid quadrupole time-of-flight mass spectrometry

Prolonged, extensive exposure to asphalt fume has been associated with several adverse health effects. Inhaled polycyclic aromatic hydrocarbons (PAHs) from asphalt fume exposure have been suspected of inducing such effects. In this study, a bioanalytical method was proposed and evaluated to identify and quantify benzo[a]pyrene and its hydroxy-metabolites. This method is based on coupling a microflow liquid chromatography (LC) to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometry (Q-TOFMS). In the experiment, thirty-two B6C3FI mice were exposed to asphalt fume in a whole body inhalation chamber for 10 days (4 h day(-1)) and twelve other mice were used as controls. The asphalt fume was generated at 180 degrees C and the concentrations in the animal exposure chamber ranged 175-182 mg m(-3). Benzo[a]pyrene and its metabolites of 3-hydroxybenzo[a]pyrene, benzo[a]pyrene-7,8-dihydrodiol(+/-), benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide(+/-), and benzo[a]pyrene-7,8,9,10-tetrahydrotetrol(+/-) in the urine of asphalt fume exposed mice were identified and found at 3.18 ng 100 mL(-1), 31.36 ng 100 mL(-1), 11.56 ng 100 mL(-1), 54.92 ng 100 mL(-1), and 45.23 ng 100 mL(-1) respectively. The results revealed that the urinary benzo[a]pyrene and its hydroxy-metabolites from exposed mice were at significantly higher levels (p < 0.001) than those from the control groups. Compared with several other technologies such as HPLC-UV and HPLC-fluorescence, the new method is more sensitive and selective, and it can also provide additional useful information on the structures of the metabolites. Hence, this method can be used to perform the assessment and to study the mechanisms of the adverse health effects. The fragmentation patterns established in this study can also be used to identify and quantify PAH metabolites in other biological fluids.     

Immunochemical detection of metabolites of parent and nitro polycyclic aromatic hydrocarbons in urine samples from persons occupationally exposed to diesel exhaust

An immunochemical assay was developed for the detection of metabolites excreted in urine as a result of occupational exposure to PAHs and nitro-PAHs. These metabolites were analyzed in a competitive ELISA, using an existing antibody (4D5) that has been developed against 6-aminobenzo[a]pyrene coupled to bovine serum albumine (B[a]P-BSA). A solid phase extraction (SPE) work-up procedure was optimized by dilution of pooled urine samples from rats exposed to 1-NP, in human urine. The application was validated by comparison of test results with urinary excreted 1-hydroxypyrene (1-OH-P) levels in a study among 28 railroad workers. Excretion of urinary metabolites was determined over two consecutive workdays. The 24 h average excretion of metabolites as determined in the immunoassay was not related to levels of particulate matter in the breathing zones of workers, not to 1-OH-P excretion levels of the day of urine collection. However, it was significantly associated with the personal dust exposure of the day before (P<0.0001), suggesting slow excretion of urinary metabolites. Smoking habits caused minor interference (P<0.1).     

Simultaneous determination of MK-0767 and seven metabolites in rat urine using liquid chromatography/tandem mass spectrometry

MK-0767, 5-[2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide (I, Table 1), is a dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist previously studied for the treatment of type 2 diabetes and dyslipidemia. To support further toxicological studies in one of the animal species used in chronic testing of I, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantification of I and seven metabolites in rat urine was developed and validated. In this method, urine samples were diluted with acetonitrile/methanol (50:50, v/v) and injected directly onto the column of an LC system. Detection was achieved by MS/MS using a turbo ion spray probe monitoring precursor --> product ion combinations in selected reaction monitoring (SRM) mode. The linear range for I and three metabolites was 0.8-800 ng/mL, and 8-8000 ng/mL for four other metabolites found to be present in urine at higher concentrations than I. Intra-day and inter-day variation using this method were < or = 13.0%. The method exhibited good linearity, reproducibility, specificity and sufficient sensitivity when used for the analysis of rat urine samples. Concentrations of I and its major metabolites in rat urine were determined in samples collected between 0-24 h after dosing on the last day of administration of nine daily oral doses to three male (1000 mg/kg/day) and three female (300 mg/kg/day) Sprague-Dawley rats. The urinary concentrations of I and its metabolites were similar in male and female rats. The average concentrations of I were 0.51 and 0.33 microg/mL in male and female rats, respectively. Concentrations of four of the seven metabolites quantified were 6- to 45-fold higher than those of I. The most abundant metabolite, with concentrations of 24.2 and 13.3 microg/mL in male and female rat urine, respectively, was a methyl sulfoxide derivative formed by oxidative cleavage of the thiazolidinedione ring, followed by S-methylation and oxidation of the sulfide intermediate.     

Biological activities of phthalocyanines. XII: Synthesis tumor uptake and biodistribution of 14C-labeled disulfonated and trisulfonated gallium phthalocyanine in C3H mice

The biodistribution and metabolism of 14C-labeled disulfonated and trisulfonated gallium phthalocyanine (Ga-PcS) was studied in radiation-induced fibrosarcoma tumor-bearing C3H mice. The [14C]Ga-PcS compounds were prepared via the condensation of [14C]phthalic acid and sulfophthalic acid in the presence of gallium chloride and characterized by their spectroscopic and chromatographic properties. The tissue concentrations of the dyes was measured by scintillation counting of the 14C and by extraction and fluorescence measurements. Elevated dye levels were found in the liver, lungs, kidneys and spleen as well as in the tumor. Lower sulfonation of Ga-PcS favored liver and spleen uptake whereas higher dye sulfonation resulted in greater kidney uptake. Both dyes showed high tumor uptake with peak concentrations exceeding those of most tissues except for the liver in the case of Ga-PcS2. The highest tumor uptake was observed with Ga-PcS3. Both dyes were slowly excreted from the body. The liver-feces pathway was favored in the case of Ga-PcS2 with high activities persisting in the liver, even after 21 days. The Ga-PcS3 was preferentially excreted via the kidney-urine pathway. High performance liquid chromatography analysis of the liver and tumor extracts of [14C]Ga-PcS3-treated animals did not reveal desulfonation of the dye. However, urine analysis showed the presence of radioactive metabolites lacking the characteristic phthalocyanine absorption.     

In vivo biotransformation of 17 alpha-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry

The in vivo phase I biotransformation of 17 alpha-methyltestosterone in the horse leads to the formation of a complex mixture of regio- and stereoisomeric C(20)O(2), C(20)O(3) and C(20)O(4) metabolites, excreted in urine as glucuronide and sulphate phase II conjugates. The major pathways of in vivo metabolism are the reduction of the A-ring (di- and tetrahydro), epimerisation at C-17 and oxidations mainly at C-6 and C-16. Some phase I metabolites have been identified previously by positive ion electron ionisation capillary gas chromatography/mass spectrometry (GC/EI + MS) mainly from the characteristic fragmentation patterns of their methyloxime-trimethylsilyl ether (MO-TMS), enol-TMS or TMS ether derivatives. Following oral administration of 17 alpha-methyltestosterone to two castrated thoroughbred male horses, the glucuronic acid conjugates excreted in post-administration urine samples were selectively hydrolysed by E. coli beta-glucuronidase enzymes. Unconjugated metabolites and the steroid aglycones obtained after enzymatic deconjugation were isolated from urine by solid-phase extraction, derivatised as MO-TMS ethers and analysed by GC/EI + MS. In addition to some of the known metabolites previously identified from the characteristic mass spectral fragmentation patterns of 17 alpha-methyl steroids, some isobaric compounds exhibiting a diagnostic loss of 103 mass units from the molecular ions with subsequent losses of trimethylsilanol or methoxy groups and an absence of the classical D-ring fragment ion were detected. From an interpretation of their mass spectra, these compounds were identified as 17-hydroxymethyl metabolites, formed in vivo in the horse by oxidation of the 17-methyl moiety of 17 alpha-methyltestosterone. This study reports on the GC/EI + MS identification of these novel 17-hydroxymethyl C(20)O(3) and C(20)O(4) metabolites of 17 alpha-methyltestosterone excreted in thoroughbred horse urine.     

Combination therapeutic effect of cisplatin along with Solanum trilobatum on benzo(a)pyrene induced experimental lung carcinogenesis

Lung cancer is one of the leading causes of cancer death in the world and is notoriously difficult to treat effectively. In the present study, male Swiss albino mice were divided into five groups of six animals each: group I animals received corn oil orally and served as a control; group II cancer-induced animals received benzo(a)pyrene (B[a]P) (50 mg kg(-1) bodyweight dissolved in corn oil, orally) twice weekly for four successive weeks; group III cancer-bearing animals (after 12 weeks of induction) were treated with cisplatin (6 mg kg(-1) bodyweight, i.p.) once weekly for 4 weeks; group IV cancer-bearing animals were treated with cisplatin along with Solanum trilobatum (300 mg kg(-1) bodyweight) orally once weekly for 4 weeks; and group V animals constituted the drug control treated with cisplatin along with S. trilobatum. The serum, lung and liver were investigated biochemically for aryl hydrocarbon hydroxylase, gamma-glutamyl transpeptidase, 5'-nucleotidase, lactate dehydrogenase (LDH) and protein-bound carbohydrate components (hexose, hexosamine and sialic acid). These enzyme activities were increased significantly in cancer-bearing animals compared with control animals. The elevation of these in cancer-bearing animals was indicative of the persistent deteriorating effect of B[a]P in cancer-bearing animals. Our data suggest that cisplatin, administered with S. trilobatum, may extend its chemotherapeutic effect through modulating protein-bound carbohydrate levels and marker enzymes, as they are indicators of cancer. The combination of cisplatin with S. trilobatum could effectively treat the B[a]P-induced lung cancer in mice by offering protection from reactive oxygen species damage and also by suppressing cell proliferation.     

Glutathione and methylation of inorganic arsenic in hamsters

The effect of giutathione (GSH) concentrations in livers and kidneys of hamsters on the toxicity and methylation of arsenite in these animals was studied. No significant changes in hepatic and renal GSH concentrations were observed after a single arsenite administration (5 mg As kg^?1, p.o.). When buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, was given (4 mmol kg^?1, i.p.) two hours before administration of arsenite, hepatic and renal GSH concentrations were more severely and persistently depressed than in the case of BSO administration not followed by arsenite. Hamsters treated with BSO plus arsenite suffered from severe nephrotoxicity (acute renal failure) characterized by increases in plasma creatinine and urea nitrogen and by proximal tubular necrosis. Concurrently, transient hepatotoxicity was observed in the BSO plus arsenite group. Neither arsenite alone nor BSO alone produced liver or kidney injury. The BSO plus arsenite-treated animals excreted in the urine only 3.5% of the arsenic dose during the 72 h period after administration of arsenite, probably because of a decrease in urine volume caused by kidney injury, whereas the arsenite-only group excreted 27%. In addition, BSO pretreatment influenced the relative proportion of arsenic metabolites excreted in the urine during the first 24 h after administration. Urinary metabolites in the BSO plus arsenite group were predominantly inorganic arsenic. These results suggest that GSH provides protection against arsenic toxicity.     

HPLC–MS Profiling and Structural Identification of [14C]-Diclofenac Metabolites in Mouse Bile

Biliary metabolites present at 6 h post-dose following a single oral dose of [¹⁴C]-diclofenac (10 mg kg^?1) to male bile duct-cannulated C57BL/6 J mice were profiled and identified. Over the 6 h duration of the study ~19.5 % of the administered radioactivity was excreted into the bile as either [¹⁴C]-diclofenac or metabolites. When profiled using HPLC with online radiodetection, the presence of at least 13 radiolabelled components was indicated. These compounds were shown, by consecutive reaction mass spectrometry, to comprise a range of hydroxylated metabolites conjugated to either taurine, glucose and/or glucuronic acid. Both phenolic and acylglucuronide-containing metabolites were observed. The confirmation of the presence of these glucuronide conjugates in mouse bile may have important consequences in the light of emerging theories concerning the role of bacterial glucuronidases for the GI-tract toxicity of NSAIDs such as diclofenac.     

Sensitive and selective determination of metabolically formed trans-dihydrodiols and phenols of benzo[a]pyrene in water and urine samples by HPLC with amperometric detection

A method has been developed to separate hydroxylated metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene, i. e. trans-4,5-, 7,8-, 9,10-dihydrodiol and 1-, 3-, 7-, and 9-phenol, by HPLC with amperometric detection employing an isocratic methanol/water eluent (70:30, v/v) containing 0.5 g/L sulfuric acid and 1 g/L lithium perchlorate. Compared with the usually applied fluorescence (λ_ex = 265 nm, λ_em = 460 nm) and ultraviolet (λ = 265 nm) detection, the amperometric technique is about 2–12 times more sensitive for the determination of all metabolites investigated. The method was applied to the determination of the seven metabolites of benzo[a]pyrene in different water samples and in urine after solid-phase extraction (SPE). The results obtained by HPLC with amperometric detection after SPE enrichment from an aqueous extract of a soil sample and from the urine of a rat intragastrically treated with benzo[a]pyrene agreed well with the values determined with fluorescence and/or UV detection.     

Urinary metabolites of cannabidiol in dog, rat and man and their identification by gas chromatography-mass spectrometry

Urinary metabolites of cannabidiol (CBD), a non-psychoactive cannabinoid of potential therapeutic interest, were extracted from dog, rat and human urine, concentrated by chromatography on Sephadex LH-20 and examined by gas chromatography-mass spectrometry as trimethylsilyl (TMS), [2H9]TMS, methyl ester-TMS and methyloxime-TMS derivatives. Fragmentation of the metabolites under electron-impact gave structurally informative fragment ions; computer-generated single-ion plots of these diagnostic ions were used extensively to aid metabolite identification. Over fifty metabolites were identified with considerable species variation. CBD was excreted in substantial concentration in human urine, both in the free state and as its glucuronide. In dog, unusual glucoside conjugates of three metabolites (4"- and 5"-hydroxy- and 6-oxo-CBD), not excreted in the unconjugated state, were found as the major metabolites at early times after drug administration. Other metabolites in all three species were mainly acids. Side-chain hydroxylated derivatives of CBD-7-oic acid were particularly abundant in human urine but much less so in dog. In the latter species the major oxidized metabolites were the products of beta-oxidation with further hydroxylation at C-6. A related, but undefined pathway resulted in loss of three carbon atoms from the side-chain of CBD in man with production of 2"-hydroxy-tris,nor-CBD-7-oic acid. Metabolism by the epoxide-diol pathway, resulting in dihydro-diol formation from the delta-8 double bond, gave metabolites in both dog and human urine. It was concluded that CBD could be used as a probe of the mechanism of several types of biotransformation; particularly those related to carboxylic acid metabolism as intermediates of the type not usually seen with endogenous compounds were excreted in substantial concentration.     

Characterization of [6]-gingerol metabolism in rat by liquid chromatography electrospray tandem mass spectrometry

[6]-Gingerol is a structural analog of capsaicin, an agonist of the transient receptor potential channel vanilloid 1, which is known to have therapeutic properties for the treatment of pain and inflammation. A selective and sensitive quantitative method for the determination of [6]-gingerol by HPLC-ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray tandem mass spectrometry. The chromatographic separation was achieved using a Thermo 100 × 2.1 mm C(8) column combined with an isocratic mobile phase composed of acetonitrile, water and formic acid (80:20:0.1) at a flow rate of 250 μL/min. The mass spectrometer was operating in SRM mode and an analytical range set at 20-5000 ng/mL was used to construct a calibration curve in rat plasma. The interbatch precision (%CV) and accuracy (%NOM) observed were 2.9-10.8% and 98.1-102.1% in rat plasma. Similarly, precision and accuracy in rat liver microsomal suspension were also evaluated at nominal concentrations of 1, 25 and 100 μm; the precision (%CV) was <3.4% and the accuracy (%NOM) observed ranged from 89.7 to 109.4%. An in vitro metabolic stability study using rat liver microsomes was performed to determine intrinsic clearance of [6]-gingerol. The results show slow degradation with a T(1/2) of 163 min and relatively low intrinsic clearance suggesting that phase I metabolism may not be a major contributor of the drug clearance. Further analyses were performed to characterize in vitro and in vivo metabolites. Three main phase I metabolites and four phase II metabolites were identified by HPLC-MS/MS and HPLC-MSD TOF. However, the results suggest that glucuronidation of hydroxylated [6]-gingerol is the primary metabolite excreted in rat urine.     

固相萃取-液相色谱-串联质谱法同时测定尿液中9种多环芳烃代谢物

建立了固相萃取-液相色谱-串联质谱同时测定尿中2-羟基萘、1-羟基萘、2-羟基芴、3-羟基菲、1-羟基芘等9种多环芳烃代谢物的液相色谱-串联质谱测定方法。尿样中结合态的多环芳烃代谢物在β-葡萄糖苷酸酶-芳基硫酸酯酶缓冲液(pH 5.0)作用下,于37℃水浴中避光水解4 h后,以C18固相萃取小柱富集、净化,以甲醇洗脱,采用Waters Symmetry C18色谱柱,流动相为乙腈-0.2%氨水(72∶27,V/V)等度淋洗分离后进入质谱测定。在喷雾电压4 kV,毛细管温度300℃下,以3-羟基菲13C为内标,采用SRM模式负离子扫描方式测定,内标法定量。9种多环芳烃代谢物在尿中的线性范围为0.90～100μg/L;相关系数为0.9970～0.9990;回收率为79.0%～119.8%;相对标准偏差为4.3%～12.4%;检出限为0.04～0.90μg/L;结果表明,本方法可用于尿中9种多环芳烃代谢物的测定。     

Control system for detection of the illegal use of naturally occurring steroids in calves

Within the scope of the National Plan for Hormone Control in The Netherlands, a study was performed to develop a system for control of the illegal use of three naturally occurring hormones [oestradiol-17 beta (E2-17 beta), testosterone (T), progesterone (P)] for fattening purposes in animal production. Using a specific high-performance liquid chromatographic-radioimmunoassay method, reference values were established for concentrations of E2-17 beta, T and P and some of their metabolites in blood plasma and urine from untreated male and female veal calves. E2-17 beta levels of both male and female calves were less than 0.01 microgram/l in blood plasma and less than 0.2 microgram/l in urine. For male veal calves levels of T and epitestosterone (epiT) in blood plasma and urine varied widely. The P levels were less than 0.1-0.3 micrograms/l in blood plasma and less than 0.6-10 micrograms/l in urine from both male and female calves. To investigate the effect of anabolic treatment on the hormone levels in plasma and excreta, male veal calves were injected, subcutaneously into the dewlap, with a solution containing 20 mg of E2-17 beta benzoate and 200 mg of T propionate in 5 ml of arachis oil. Only the levels of E2-17 beta and E2-17 alpha in blood plasma and excreta were elevated until about one week after injection, compared with the untreated control calves and the reference values. T and epiT levels were similar in plasma and excreta from both untreated and treated animals.     

Quantification of monohydroxy-PAH metabolites in urine by solid-phase extraction with isotope dilution-GC–MS

For measurement of biomarkers from polycyclic aromatic hydrocarbon (PAH) exposure, an analytical method is described quantifying hydroxylated PAH (OH-PAH) in urine samples. This method determined monohydroxy metabolites of naphthalene, fluorene, phenanthrene, fluoranthene, pyrene, chrysene, benzo[c]phenanthrene, and benz[a]anthracene. The sample preparation consisted of enzymatic hydrolysis, solid-phase extraction and derivatization with a silylating reagent. Five carbon-13 labeled standards were used for isotope dilution. Analytes were separated by gas chromatography (GC) and quantified with high-resolution mass spectrometry (HRMS). This method produced good recoveries (41-70%), linearity, and specificity. Data were corrected for blank levels from the naphthalene, fluorene, and phenanthrene metabolites. Method detection limits ranged from 2 ng L(-1) for 1-hydroxypyrene to 43.5 ng L(-1) for 1-hydroxynaphthalene. Using quality control charts from two urine pools, the method can be readily applied to biomonitoring PAH exposure.     

Sensitive and selective determination of metabolically formed trans-dihydrodiols and phenols of benzo[a]pyrene in water and urine samples by HPLC with amperometric detection

A method has been developed to separate hydroxylated metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene, i. e. trans-4,5-, 7,8-, 9,10-dihydrodiol and 1-, 3-, 7-, and 9-phenol, by HPLC with amperometric detection employing an isocratic methanol/water eluent (70:30, v/v) containing 0.5 g/L sulfuric acid and 1 g/L lithium perchlorate. Compared with the usually applied fluorescence (λ_ex = 265 nm, λ_em = 460 nm) and ultraviolet (λ = 265 nm) detection, the amperometric technique is about 2–12 times more sensitive for the determination of all metabolites investigated. The method was applied to the determination of the seven metabolites of benzo[a]pyrene in different water samples and in urine after solid-phase extraction (SPE). The results obtained by HPLC with amperometric detection after SPE enrichment from an aqueous extract of a soil sample and from the urine of a rat intragastrically treated with benzo[a]pyrene agreed well with the values determined with fluorescence and/or UV detection. Received: 20 December 1996 / Revised: 10 March 1997 / Accepted: 30 April 1997     

Speciation of metabolites of selenate in rats by HPLC-ICP-MS

The metabolic pathway for and metabolites of selenium (Se) administered intravenously to rats in the form of selenate at a dose of 0.3 mg Se kg-1 body weight were studied by speciating Se in the bloodstream, liver and urine by HPLC-inductively coupled argon plasma mass spectrometry. Selenate was not taken up by red blood cells (RBCs) and disappeared from the bloodstream much faster than selenite, without any change in its chemical form before it disappeared from the plasma. Selenium excreted into the urine after the administration of selenate showed different patterns from those of selenite in both amounts and chemical forms. With the selenate group, the concentration of Se in urine was highest at 0-6 h and the chemical species of Se was selenate at 0-6 h; thereafter a monomethylselenol-related Se compound (MMSe*) and trimethylselenonium ions (TMSe) appeared, selenate not being excreted after 6 h. On the other hand, in the selenite group, the concentration of Se peaked at 6-12 h, and the chemical species of Se were MMSe* and TMSe. Selenate was reduced in vitro on incubation in either a liver homogenate or supernatant fraction, although much more slowly than in the whole body. Selenate was not reduced by glutathione or dithiothreitol. The results suggest that in contrast to selenite, which is taken up by and reduced in RBCs, and then transferred to the liver, approximately 20% of the selenate administered to rats was excreted into the urine without any change in its chemical form with the present dose, and the major portion of selenate was taken up by the liver, reduced and then utilized for the synthesis of selenoproteins or excreted into the urine after being methylated.     

ENHANCED GROWTH AND EXPERIMENTAL METASTASIS OF CHEMICALLY INDUCED TUMOR IN ULTRAVIOLET IRRADIATED SYNGENEIC MICE

Abstract— Recent studies have shown that ultraviolet (UV) irradiation induces a systemic effect which enhances subsequent tumor induction by benzo[a]pyrene in a manner which is dependent on the dose of benzo[a]pyrene. The present study was designed to test whether UV-B irradiation renders mice susceptible to subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene. The sources of UV-B irradiation were banks of 6 Westinghouse FS-40 sunlamps, situated 20 cm above the mouse cages. Female BALB/cAnNHsd received five 30-min dorsal UV-B radiation treatments per week for 12 weeks, resulting in a total dose of approx. 6.4 × 10⁵ J m^-2. Two to seven days after termination of UV treatments, syngeneic regressor tumor cells (BP2) induced by benzo[a]pyrene were injected subcutaneously or intravenously into irradiated mice and unirradiated controls. By 38 days post subcutaneous implantation, 24/30 and 3/30 BP2 implants were detectable in the irradiated and unirradiated mice, respectively. Ultraviolet irradiated mice were also unable to reject lung colonies resulting from intravenous administration of BP2 cells, although they were rejected by unirradiated mice. The mean number of lung colonies per mouse was 16- to 35-fold greater in UV irradiated mice than in unirradiated controls, at 14 to 17 days post injection. Thus, UV irradiation rendered mice, with no known exposure to benzo[a]pyrene, susceptible to a subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene.     

Analysis of some metabolites of T-2 toxin, diacetoxyscirpenol and deoxynivalenol by thermospray high-performance liquid chromatography-mass spectrometry

Detection and identification of mycotoxin metabolites is a very challenging task. In order to achieve adequate sensitivity and specificity an analytical technique must overcome serious matrix interferences. Gas chromatography-mass spectrometry (GC-MS) which has the sensitivity and specificity to detect and identify mycotoxin metabolites requires hydrolysis of conjugated metabolites as well as derivatization. Thermospray high-performance liquid chromatography-mass spectrometry (HPLC-MS) offers the sensitivity, specificity, and structural information to detect and identify some mycotoxin metabolites in fecal and urine samples without derivatization. The mycotoxins evaluated in this study include deoxynivalenol (DON), T-2 toxin, and diacetoxyscirpenol. The de-epoxy and hydroxy metabolites of each toxin and the glucuronide conjugate of DON were isolated, extracted, and analyzed to detect their occurrence in animals. The thermospray mass spectra of the toxins showed an [M + H]+ ion and numerous structurally significant fragment ions in the positive ion detection mode. Negative ion detection exhibited primarily [M + acetate]- cluster ions with less fragmentation than observed by positive ion detection. The operation of the interface in the filament-on mode greatly increased the sensitivity in both positive and negative ion detection mode. Detection limits of 50-500 pg injected on column are obtained for these toxins and their metabolites using multiple ion detection. The urine and fecal extracts from rats, hens, and cows did not interfere with the HPLC-MS analysis for the specific metabolites or the glucuronide conjugate.