Nitrogen inputs best predict farm field nitrate leaching in the Willamette Valley, Oregon.

Nitrate leaching is an important yet difficult to manage contribution to groundwater and surface water contamination in agricultural areas. We examine 14 farm fields over a four year period (2014-2017) in the southern Willamette Valley, providing 53 sets of annual, field-level agricultural performance metrics related to nitrogen (N), including fertilizer inputs, crop harvest outputs, N use efficiency (NUE), nitrate-N leaching and surplus N. Crop-specific nitrate-N leaching varied widely from 10 kg N ha-1yr-1 in hazelnuts to >200 kg N ha-1yr-1 in peppermint. Averaging across all sites and years, most leaching occurred during fall (60%) and winter (32%). Overall NUE was 57%. We used a graphical approach to explore the relationships between N inputs, surplus, crop N harvest removal and NUE by crop type. The blueberry site had high inputs and surplus, peppermint had high inputs but also high crop N removal and NUE and thus lower surplus, and most wheat crops had high NUE and evidence of using soil N. Annual N surplus was not well correlated with leaching, and leaching varied more by crop type and inputs. Grass seed and hazelnuts, which are dominant crop types in the southern Willamette Valley, were intermediate in terms of NUE, leaching and surplus. Of all performance metrics, N input was most closely aligned with field-level crop N harvest and nitrate leaching, therefore optimizing N inputs may well inform local efforts to reduce groundwater nitrate contamination.

Structure, genetic mapping, and function of the cytochrome P450 3A37 gene in the turkey (Meleagris gallopavo).

Cytochromes P450 (P450 for protein; CYP for gene) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. Through oxidation reactions, these enzymes are often responsible for the toxic and carcinogenic effects of natural food-borne toxicants, such as the mycotoxin aflatoxin B1 (AFB1). Previous studies in our laboratory have shown that the extreme sensitivity of turkeys to AFB1 is in part explained by efficient hepatic P450-mediated epoxidation to the toxic and reactive metabolite the exo-AFB1-8,9-epoxide (AFBO). Using 3'-5'-rapid amplification of cDNA ends (RACE), we amplified CYP3A37 from turkey liver RNA, the E. coli-expressed protein which efficiently epoxidates AFB(1). Turkey CYP3A37 has an ORF of 1512 bp, and the protein is predicted to be 504 amino acids with 97% homology to chicken CYP3A37. The turkey gene is organized into 13 exons and 12 introns. A single nucleotide polymorphism in the 11th intron was used to assign CYP3A37 to turkey linkage group 10 (corresponding to chicken chromosome 14, GGA14). Because of the important role of P450s in the extreme sensitivity of turkeys to the toxic effects of AFB(1), this study will contribute to the identifying allelic variants of this important gene in poultry.

Butylated hydroxytoluene chemoprevention of aflatoxicosis - effects on aflatoxin B(1) bioavailability, hepatic DNA adduct formation, and biliary excretion.

The extreme sensitivity of turkeys to aflatoxin B(1) (AFB(1)) is associated with efficient hepatic cytochrome P-450 (P450)-mediated bioactivation, and deficient glutathione S-transferase (GST) mediated detoxification. Butylated hydroxytoluene (BHT) protects against AFB(1) toxicity in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB(1) bioactivation. To test whether dietary BHT alters hepatic AFB(1)-DNA adduct formation, excretion, and bioavailability of AFB(1)in vivo, turkeys were given diets with BHT (4000ppm) for 10 days, given a single oral dose of [(3)H]-AFB(1) (0.05microg/g; 0.02microCi/g), then sampled at intervals up to 24h. Radiolabel in serum, red blood cells, liver, and breast meat was frequently lower in BHT-treated compared to control. Hepatic AFB(1)-DNA adducts in BHT-treated turkeys were significantly lower at 12 and 24h. BHT-fed birds had significant higher bile efflux, though biliary radiolabel excretion was not different from control. The amount of aflatoxin M(1) (AFM(1)) excreted in the bile was lower than in control, but BHT had no effect on the biliary excretion of AFB(1), aflatoxin Q(1) or glucuronide and sulfate conjugates. Thus, the chemopreventive properties of BHT may also occur through a reduction in AFB(1) bioavailability in addition to inhibition of bioactivation.

Structure and genetic mapping of the Cytochrome P450 gene (CYP1A5) in the turkey (Meleagris gallopavo).

Cytochromes P450 (P450) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. The recently cloned P450 gene (CYP1A5) encodes the primary protein responsible for epoxidation of aflatoxin B(1) (AFB(1)) in the turkey, an animal extremely sensitive to this mycotoxin. Hypersensitivity of turkeys to AFB(1) was first demonstrated by association with 'Turkey X Disease' which caused widespread deaths of turkeys and other poultry throughout Europe in the 1960s, later shown to be caused by AFB(1)-contaminated feed. In this study, comparative genomic approaches were used to selectively amplify and sequence the introns and 3' flanking region of CYP1A5. The structure of the CYP1A5 gene in the turkey is shown to be equivalent to that of the human CYP1A genes with seven exons of 38, 858, 127, 90, 124, 87 and 307 bp, respectively, and six introns. A single nucleotide polymorphism (SNP) in the 3' UTR was used to assign CYP1A5 to turkey linkage group M16 (equivalent to chicken chromosome 10). The results of this study provide the framework for identifying allelic variants of this biochemically important P450 gene in poultry.

Effects of dietary butylated hydroxytoluene on aflatoxin B1-relevant metabolic enzymes in turkeys.

We have shown previously that the extreme sensitivity of turkeys to aflatoxin B(1) (AFB(1)) is due to a combination of efficient AFB(1) activation by cytochrome P450s (CYPs) 1A and deficient detoxification by glutathione S-transferases (GSTs). Phenolic antioxidants such as butylated hydroxytoluene (BHT) have been shown to be chemoprotective in some animal models due, in part, to modulation of AFB(1)-relevant phase I and/or phase II activities, and we wished to determine whether BHT has a similar effect in turkeys. Ten-day-old male turkeys were maintained on diets amended with 1000 or 4000 ppm of BHT for 10 days, then sampled. Hepatic microsomal CYP 1A activity as well as conversion of AFB(1) to the putative toxic metabolite, the exo-AFB(1)-8,9-epoxide (AFBO), were significantly lower compared with control. Conversely, dietary BHT significantly increased activities of several isoforms of hepatic cytosolic GST, as well quinone oxidoreductase (QOR). Western immunoblotting confirmed that dietary BHT increased expression of homologues to rodent GST isoforms Yc1, Yc2 and Ya. There was, however, no observable BHT-related increase in GST-mediated specific conjugation with microsomally-generated AFBO. In total, our data indicates that dietary BHT modulates a variety of AFB(1)-relevant phase I and phase II enzymes, while having no measurable effect towards specific AFB(1) detoxification by GST.

Inhibition of human cytochrome P450 2E1 by nicotine, cotinine, and aqueous cigarette tar extract in vitro.

Cigarette smoke is a complex mixture containing, among other chemicals, pyridine alkaloids and N-nitrosamines. Carcinogenic tobacco-specific N-nitrosamines, N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are both activated by cytochrome P450 (CYP) 2E1 in rats. Previous reports indicate that nicotine and the main nicotine metabolite, cotinine, reduce the mutagenicity of both NNK and NDMA in Salmonella typhimurium. To study the mechanism of this effect, we examined inhibition of CYP 2E1 activity, as assessed by p-nitrophenol (pNP) hydroxylation, by nicotine, cotinine, and an aqueous cigarette tar extract (ACTE) in human 2E1-expressing microsomes. At all substrate concentrations (0-1.25 mM) nicotine was a significantly more potent inhibitor of CYP 2E1 activity compared to cotinine. Estimated Ki values for nicotine and cotinine (both at 10 mM) were 13 mM (2 mg/ml) and 308 mM (54 mg/ml) respectively. The Ki for ACTE was 0.2 mg/ml at a concentration of 0.32 mg/ml. This rank order for inhibition was also seen when the data was expressed as IC(50). When compared on a mass/vol basis, ACTE was a significantly more potent CYP 2E1 inhibitor relative to nicotine and cotinine. Double-reciprocal plots indicated that nicotine and ACTE inhibited by a competitive, while cotinine inhibited CYP 2E1 by an uncompetitive mechanism. Although the contribution of nicotine to ACTE-mediated 2E1 inhibition is probably modest, pyridine alkaloid-mediated CYP 2E1 inhibition is a possible mechanism for the observed inhibition of NNK and NDMA mutagenicity by nicotine and cotinine in vitro.

Metabolism of aflatoxin B1 by normal human bronchial epithelial cells.

Aflatoxin B, (AFB1) is a potent hepatocarcinogen in animal models and a suspected carcinogen in humans. High concentrations of AFB, have been found in respirable grain dusts, and may therefore be a risk factor for human lung cancer in certain occupations. To study the potential for AFB, activation in human lung, cytochrome P-450 (CYP)-mediated activation and glutathione S-transferase (GST)-mediated detoxification of AFB1 were examined in cultured normal human bronchial epithelial (NHBE) cells. Cells were exposed to 0. 15 microM or 1.5 microM AFB, for 48 h and media was collected for metabolite analysis by high-performance liquid chromatography (HPLC). At 0. 15 microM, AFB1 was metabolized only to the detoxified metabolite aflatoxin Q1 (AFQ1). At 1.5 microM AFB1, both aflatoxin M1 (AFM1), and AFQ1 were produced. Cells pretreated with 50 degrees M 3-methylcholanthrene (3MC), a CYP 1A inducer, for 72 h prior to 0.15 microM AFB1, produced the activated AFB1 8,9-epoxide (AFBO). Similarly, microsomes prepared from 3MC-pretreated cells formed AFBO, but microsomes from noninduced cells did not. While AFB1-DNA adducts were not detected at low AFB1 concentrations in untreated NHBE, 3MC induction caused the production of AFB1-DNA adducts at 0.015 and 0.15 microM AFB1. Western immunoblots showed that the primary CYP isoforms responsible for AFB1 activation in the liver, 1A and 3A4, to be constitutively expressed in NHBE cells. Expression of CYP 1A was significantly increased in 3MC-pretreated cells, while CYP 3A4 expression increased slightly, but not to the extent of the 1A isoforms. The principal AFBO detoxifying enzyme, glutathione S-transferase (GST), was constitutively expressed in NHBE cells, and was increased approximately twofold by 3MC pretreatment. Cytosolic fractions from neither control nor 3MC-induced NHBE had measurable AFBO conjugating activity, indicating that these cells may lack AFB1-relevant GST activity. From these data, it appears that NHBE cells activate AFB1 inefficiently, but possess CYPs reportedly responsible for metabolism of AFB1. These data support earlier findings showing modest CYP-mediated AFB1 activation in human airways, but indicate that exposure to polycyclic aromatic hydrocarbons (PAHs), such as 3MC, which induce CYP(s) that specifically activate AFB1 may increase the harmful effects of AFB1 exposures in human airways.

Biochemical basis for the extreme sensitivity of turkeys to aflatoxin B(1).

Poultry are the most susceptible food animal species to the toxic effects of the mycotoxin aflatoxin B(1) (AFB(1)). Feed contaminated with even small amounts of AFB(1) results in significant adverse health effects in poultry. The purpose of this study was to explain the biochemical mechanism(s) for this extreme sensitivity. We measured microsomal activation of AFB(1) to the AFB(1)-8,9-epoxide (AFBO), the putative toxic intermediate, as well as cytosolic glutathione S-transferase (GST)-mediated detoxification of AFBO, in addition to other hepatic phase I and phase II enzyme activities, in 3-week-old male Oorlop strain turkeys. Liver microsomes prepared from these turkeys activated AFB(1) in vitro with an apparent K(m) of 109 microM and a V(max) of 1.25 nmol/mg/min. Preliminary evidence for the involvement of cytochromes P450 (CYP) 1A2 and, to a lesser extent, 3A4 for AFB(1) activation was assessed by the use of specific mammalian CYP inhibitors. The possible presence of avian orthologues of these CYPs was supported by activity toward ethoxyresorufin and nifedipine, as well as by Western immunoblotting using antibodies to human CYPs. Cytosol prepared from turkey livers exhibited GST-mediated conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB), but at a much lower rate than that observed in other species. Western immunoblotting indicated the presence of alpha and sigma class GSTs and another AFB(1)-detoxifying enzyme, AFB(1)-aldehyde reductase (AFAR). Turkey liver cytosol also had quinone oxidoreductase (QOR) activity. Importantly, cytosol exhibited no measurable GST-mediated detoxification of microsomally activated AFB(1), indicating that turkeys are deficient in the most crucial AFB(1)-detoxification pathway. In total, our data indicate that the extreme sensitivity of turkeys to AFB(1) may be attributed to a combination of efficient AFB(1) activation and deficient detoxification by phase II enzymes, such as GSTs.

Comparative DNA cross-linking by activated pyrrolizidine alkaloids.

The toxicity and bioactivity of pyrrolizidine alkaloids (PAs), common constituents of hundreds of plant species, and in herbal remedies and folk medicines prepared thereof, are probably due to their ability to form DNA cross-linking. We investigated DNA cross-linking activity by chemically-activated PAs from four different structural classes in Madin-Darby bovine kidney (MDBK) cells and in pBR322 DNA. In cell culture, alpha,beta-unsaturated macrocyclic diester pyrroles dehydrosenecionine (DHSN), dehydroriddelliine (DHRD) and the saturated macrocyclic diester pyrrole dehydromonocrotaline (DHMO) were significantly more potent cross-linkers than the simple necine base (retronecine) and an N-oxide (indicine N-oxide; INO) as determined by alkaline elution. The proportion of total DNA cross-links that were proteinase K-resistant (DNA-DNA cross-links) induced by the various pyrroles ranged from 0.08 (DHRN) to 0.67 (DHSN). Those pyrroles that were potent cross-linkers of cellular DNA also cross-linked, in a dose-dependent manner, Bam HI-digested pBR322 DNA as assessed by a gel retardation assay. The possible functional relevance of pyrrole-DNA cross-links was determined by their ability to interrupt PCR amplification of a 1.129 kb segment of pBR322. Dehydrosenecionine completely inhibited amplification, while DHMO was of intermediate potency, while DHRN and INO had no effect. Taken together, these studies suggest that structural features, most notably the presence of a macrocyclic diester, confer potent cross-link activity to PAs. In any event, DNA-DNA cross-linking is probably biologically relevant as indicated by their interference with DNA replication.

Pyrrolizidine alkaloid plants, metabolism and toxicity.

More than 350 PAs have been identified in over 6,000 plants in the Boraginaceae, Compositae, and Leguminosae families (Table 1). About half of the identified PAs are toxic and several have been shown to be carcinogenic in rodents. PA-containing plants have worldwide distribution, and they probably are the most common poisonous plants affecting livestock, wildlife, and humans. In many locations, PA-containing plants are introduced species that are considered invasive, noxious weeds. Both native and introduced PA-containing plants often infest open ranges and fields, replacing nutritious plants. Many are not palatable and livestock avoid eating them if other forages are available. However, as they invade fields or crops, plant parts or seeds can contaminate prepared feeds and grains which are then readily eaten by many animals. Human poisonings most often are a result of food contamination or when PA-containing plants areused for medicinal purposes. This is a review of current information on the diagnosis, pathogenesis, and molecular mechanisms of PA toxicity. Additional discussion includes current and future research objectives with an emphasis on the development of better diagnostics, pyrrole kinetics, and the effects of low dose PA exposure.

Pyrrolizidine alkaloids crosslink DNA with actin.

Pyrrolizidine alkaloids (PAs) are toxic constituents of hundreds of plant species, some of which people are exposed to in herbal products and traditional remedies. The bioactivity of PAs are related, at least in part, to their ability to form DNA-protein complexes (DPC). Previous studies from our laboratory indicated a possible role for actin in PA-induced DPCs. Nuclei prepared from Madin-Darby bovine kidney (MDBK) and human breast carcinoma (MCF-7) cells were treated with the pyrrolic PAs dehydrosenecionine (DHSN) and dehydromonocrotaline (DHMO). DPCs were purified and then analyzed by Western immunoblotting. Actin was found in DPCs induced by both DHSN and DHMO, but not in those from control nuclei. Actin was also present in DPCs induced by cisplatinum and mitomycin C, two bifunctional cross-linkers. In separate experiments, DHSN and DHMO were crosslinked to a mixture of HindIII digested lambda phage with varying amounts of glutathione (GSH), cysteine, or methionine to identify the stoichiometry of competition between DNA and alternate nucleophiles for crosslink formation with pyrroles. GSH and cysteine, but not methionine, competed with lambda phage for DNA crosslinking, indicating that reduced thiols may have a role in nucleophilic reactions with pyrroles in the cell. While actin involvement in cisplatinum-induced DPCs is documented, the discovery of actin crosslinking in PA or mitomycin C-treated cells or nuclei is, to our knowledge, novel. Pyrrole-induced DPC formation with actin, a protein with structural and/or regulatory importance proteins, may be a significant mechanism for PA toxicity and bioactivity.

Aflatoxin B1 activation in human lung.

Inhalation exposure to the carcinogen aflatoxin B1 (AFB1) in certain occupations is considerable. Because circumstantial epidemiological evidence suggests that AFB1 inhalation may cause primary lung cancer, we investigated AFB1 activation by human lung microsomes. Microsomes were incubated with [3H]AFB1 (124 microM), and activation to the AFB1-8,9-epoxide was measured as the AFB1-glutathione (AFB1-GSH) conjugate by HPLC. The formation of AFB1-GSH was in the range of 0.05-0.073 fmol/mg protein/min. The role of cytochrome P450 (CYP) 3A in this activation was investigated by oxidation of nifedipine (a prototype substrate for CYP 3A), by immunoinhibition, and by immunoblot analysis. Nifedipine oxidation varied from 0.2 to 19.2 pmol/mg protein/min in microsomes from different subjects, but did not correlate with AFB1 activation. Anti-human polyclonal CYP 3A4 IgG inhibited AFB1 activation. CYP 3A isoforms were immunoestimated to be in the range of 0.01-1.90 pmol/mg protein. Neither CYP 1A2 nor associated activity was detected in the lung microsomes. These data indicate that human lung microsomes activate AFB1 to form the exo-AFB1-8,9-epoxide and that CYP(s) of the 3A subfamily may be responsible for this activity. The relatively low amount of AFB1 activation in human lung compared to that in human liver can be explained by the scarcity of CYP-containing cells in the lung. In situ AFB1 activation and resultant carcinogenic risk are distinctly possible in occupational settings where inhalation of AFB1-contaminated dusts occurs.

Pyrrolizidine alkaloid-induced DNA-protein cross-links.

Pyrrolizidine alkaloids (PAs) are potent carcinogenic and anti-mitotic compounds produced by a large number of plant species. In this study, we investigated in vitro the DNA-protein cross-linking activity of several structurally diverse PAs. The DNA cross-linked proteins induced by PAs were also isolated and characterized in mammalian cells. At 300 and 500 microM, the pyrrolic PAs (dehydrosenecionine, dehydromonocrotaline, dehydroseneciphylline, dehydroriddelliine) induced potent DNA cross-links. Protein-associated DNA cross-links accounted for approximately 50% of the total cellular DNA cross-links at 300 microM. The simple necine pyrrole dehydroretronecine induced few DNA--protein cross-links and none were detected with indicine N-oxide. The major proteins cross-linked to DNA from either PA-exposed cells or pyrrolic PA-exposed nuclei were in the molecular weight 40-60 kDa range and were primarily acidic in nature (Ca. pI 4.2-5.0). The patterns of the proteins cross-linked to DNA were similar to that induced by standard bifunctional alkylating agents mitomycin C, cisdichlorodiammine platinum(II) and nitrogen mustard. The macrocyclic pyrrole dehydrosenecionine induced DNA cross-links in pBR322 plasmid DNA with BSA as a protein target. Our data indicated that pyrrolic PAs with a macrocyclic diester such as dehydrosenecionine, dehydroseneciphylline, dehydroriddelliine and dehydromonocrotaline were more potent cross-linkers than the simple necine pyrrolic dehydroretronecine. Cross-linking potency of the PAs examined here coincides with known potency differences in animal toxicity and led us to conclude that DNA--protein cross-linking activity is probably involved in PA-related

Pharmacokinetics of the antiviral agent 3-deazaneplanocin A.

The pharmacokinetics of 3-deazaneplanocin A (c3Nep), a competitive inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase and novel antiviral agent, was investigated in female BALB/c mice. Animals were given a single intravenous dose of [3H]-c3Nep (0.1 mg/kg; 10 microCi), and blood and selected tissues were collected at various intervals thereafter for up to 72 h. The plasma concentration versus time data for c3Nep was best approximated by a two-compartment open model with first order elimination. The elimination half-life was 12.8 min, the area-under curve (AUC) was 3.38 micrograms.min.ml-1. The distribution of c3Nep into tissues was not extensive. Following 30, 120 min, and 24 h after dosing, the kidneys and the liver contained the highest amount of drug, but this amount did not exceed 1 microgram/g tissue. At these time periods, the majority of activity in the tissues represented labeled derivatives of c3Nep indicating that this compound was converted to stable metabolites. The presence of labeled conversion products in the blood confirmed that this drug is metabolized in vivo. The fact that c3Nep bound to plasma proteins in vitro may explain this drug's limited tissue distribution. The half-life and tissue distribution of c3Nep were different from those of carbocyclic 3-deazaadenosine, a related adenosine nucleoside antiviral drug and AdoHcy hydrolase inhibitor.

Comparative activation of aflatoxin B1 by mammalian pulmonary tissues.

Occupational exposures to respirable dusts contaminated with the mycotoxin aflatoxin B1 (AFB1) have been associated with an increased incidence of upper airway tumors. To investigate this possible etiology we compared the abilities of tracheal and lung S9 from rabbit, hamster and rat to activate AFB1 to mutagens in Salmonella typhimurium TA 98. The activation of AFB1 was compared to that of benzo[a]pyrene (B[a]P), a known respiratory carcinogen. These species differ in airway morphology with respect to numbers of metabolically-active non-ciliated tracheal epithelial cells. Tracheas from hamster and rabbit and lung from rabbit were active in converting AFB1 to bacterial mutagens. Trachea from hamster was more efficient in activating AFB1 to mutagens than lung, while rabbit lung was over 4 times more active in converting AFB1 to mutagens than that from trachea. In all cases, AFB1 was more mutagenic than B[a]P. The relative capabilities of trachea to activate AFB1 were in agreement with the ability of cultured tracheas from these species to form to AFB1-DNA adducts. These results demonstrate that AFB1 is activated more efficiently than B[a]P in the lung, and that the metabolic capabilities of airway epithelium to activate AFB1 are not predictable by airway morphology.

Structural influences on pyrrolizidine alkaloid-induced cytopathology.

Pyrrolizidine alkaloids (PAs), which are common constituents of hundreds of plant species around the world, have been reported to have cytotoxic, carcinogenic, antineoplastic, and genotoxic activity in vivo and in vitro. The exact mechanism of these biological toxicities is not yet clear. The ability of eight PA congeners to inhibit mitosis and induce megalocyte formation in cultured bovine kidney epithelial cells was studied to examine possible structural influences on these endpoints. Representatives of the three PA structural groups, the macrocycles (seneciphylline, senecionine, riddelliine, retrorsine, monocrotaline), open diesters (heliosupine, latifoline), and a necine base (retronecine), were cocultured for 2 hr with a NADPH-generating system and rat liver S9. Macrocyclic PAs with alpha,beta-unsaturation (seneciphylline, senecionine, riddelliine, retrorsine) showed a dose-dependent inhibition of colony formation at 50, 100, and 300 microM and induction of megalocytosis at 500 microM. Colony growth resumed 3 weeks after removal of PAs at 50 and 100 microM, and normal cellular morphology returned 5 or 6 weeks after removal of PAs at 500 microM. The saturated macrocyclic (monocrotaline) and open diesters (heliosupine, latifoline), elicited only a slight inhibition of colony formation and had no effect on cellular morphology at 500 microM. The necine base (retronecine) had no effect on either colony formation or cell morphology. Pyrrolic PAs (dehydrosenecionine, dehydromonocrotaline, dehydroretronecine) were more active in inhibition of colony formation than their parent compounds and were potent inducers of abnormal cellular morphology at 500 microM. An N-oxide metabolite, indicine N-oxide, was completely inactive. The results support previous studies showing that there are structural influences on PA-induced cytopathological effects.

Pharmacokinetics of the antiviral agent carbocyclic 3-deazaadenosine.

The pharmacokinetics of carbocyclic 3-deazaadenosine (Cc3Ado), a competitive inhibitor of S-adenosyl-L-homocysteine hydrolase and novel antiviral agent, was investigated in female BALB/c mice. Mice were dosed either orally or intravenously with a single dose of 10 mg/kg (10 microCi [3H]Cc3Ado), and blood and tissue samples were taken at selected intervals for 24 hr. The plasma concentration vs. time data for Cc3Ado was best described by a two-compartment open model with first-order elimination. The apparent half-life was 23 and 38 min, for intravenously and orally administered Cc3Ado, respectively. Depending on route, tissue concentrations of Cc3Ado reached their maximum by 120 min, and concentrations of Cc3Ado were greatest in the liver, followed by the kidney, spleen, and stomach. By 24 hr, all tissues contained a similar amount of Cc3Ado. In the plasma, one major labeled metabolite was detected, which increased in concentration over time until about 45 min after dosing. None of the plasma Cc3Ado was protein bound, as assessed by in vitro protein binding analysis. Oral Cc3Ado was about 20% bioavailable. Data from this first investigation of the pharmacokinetics of Cc3Ado indicate that this antiviral agent is rapidly distributed and eliminated from the plasma and that distribution to tissues is widespread and rapid.

Biological action of mycotoxins.

Mycotoxins are ubiquitous, mold-produced toxins that contaminate a wide variety of foods and feeds. Ingestion of mycotoxins cause a range of toxic responses, from acute toxicity to long-term or chronic health disorders. Some mycotoxins have caused outbreaks of human toxicoses, and at least one mycotoxin, aflatoxin B1, is a presumed human hepatocarcinogen. As part of a comprehensive effort to curtail the adverse health effects posed by mycotoxins, substantial research has been conducted to determine the mechanism of action of mycotoxins in animals. This review presents some of the current knowledge on the biological action of four diverse classes of mycotoxins--aflatoxin B1, tricothecenes, zearalenone, and fumonisin B1--with particular emphasis on mechanisms of action.

Immunological and neurobiochemical alterations induced by repeated oral exposure of phenol in mice.

Phenol, a major metabolite of benzene, is a potentially immunotoxic and neurotoxic substance of environmental significance. Male CD-1 mice were continuously exposed to 0, 4.7, 19.5, and 95.2 mg phenol/l in drinking water for 4 weeks. Various immune functions were evaluated and levels of selected neurotransmitters and metabolites measured in discrete brain regions. The doses of phenol did not produce any overt clinical signs of toxicity; peripheral red blood cell counts and hematocrits decreased. A dose of 95.2 mg/l suppressed the stimulation of cultured splenic lymphocytes by lipopolysaccharide, pokeweed mitogen, and phytohemagglutinin and the response in mixed lymphocyte cultures. The two high doses suppressed antibody production response to the T cell-dependent antigen (sheep erythrocytes), as determined by plaque-forming cells, and serum antibody levels. Mice treated with phenol had lower levels of neurotransmitters in several brain regions. In the hypothalamus, a major norepinephrine-containing compartment, the concentrations of norepinephrine significantly decreased by 29 and 40% in groups dosed with 19.5 and 95.2 mg/l, while dopamine concentrations decreased in the corpus striatum by 21, 26, and 35% at 4.7, 19.5 and 95.2 mg/l, respectively. Phenol also decreased 5-hydroxytryptamine in the hypothalamus, medulla oblongata, midbrain and corpus striatum. Levels of monoamine metabolites decreased in the hypothalamus (5-hydroxyindoleacetic acid), midbrain (vanillylmandelic acid), corpus striatum (vanillylmandelic acid and dihydroxyphenylacetic acid), cortex (vanillylmandelic acid), and cerebellum (dihydroxyphenylacetic acid).

DNA cross-linking in mammalian cells by pyrrolizidine alkaloids: structure-activity relationships.

Pyrrolizidine alkaloids (PAs) are common constituents of many species of flowering plants which possess carcinogenic as well as anticarcinogenic activity in vivo. Pyrrolizidine alkaloids are genotoxic in various short-term assays. The mechanisms by which these compounds exert these effects is still unclear. In this study, we characterized the ability of eight bifunctional PAs, with differing stereochemistry and functional groups, to cross-link cellular DNA in cultured bovine kidney epithelial cells. PAs representative of three major structural classes, the macrocycles (seneciphylline, riddelline, retrorsine, senecionine, monocrotaline), the open diesters (heliosupine, latifoline), and pyrrolizidine base (retronecine) were cultured for 2 hr with cells and an external metabolizing system. Every PA induced DNA cross-links which consisted primarily of proteinase-sensitive cross-links (DPC), but also to a smaller extent, DNA interstrand cross-links (ISC). None of the PAs induced detectable amounts of DNA single-strand breaks. The PAs which produced DPC and/or ISC (ranked from most potent to least) were: seneciphylline (DPC greater than ISC); riddelline (DPC greater than ISC); retrorsine (DPC greater than ISC); senecionine (DPC greater than ISC); heliosupine (DPC greater than ISC); monocrotaline (ISC = DPC); latifoline (DPC greater than ISC); and retronecine (ISC greater than DPC). Although the PAs induced DNA cross-linking to varying degrees, cell viabilities for all treatment groups were greater than 90% as determined by trypan blue dye exclusion. Since the cross-linking ability of these PAs paralleled their ability to inhibit colony formation, cross-link formation may be involved in the biological activity of these compounds. Two structural determinants of biological activity appear to be the presence of both a macrocyclic necic acid ester and an alpha,beta-unsaturated ester function since the cross-linking ability of seneciphylline, riddelline, retrorsine, and senecionine far exceeded that of monocrotaline, heliosupine, latifoline, and retronecine. In addition, the stereochemical orientation of the ester linkage was found to have no effect on biological activity.