Molecular cloning and nucleotide sequence of a new P450 gene, CYP319A1, from the cattle tick, Boophilus microplus.

We have isolated and sequenced a novel P450 gene (CYP319A1) from the cattle tick, Boophilus microplus. The CYP319A1 cDNA encodes a protein of 531 amino acids with an estimated molecular weight of 60.9k. It contains all highly conserved motifs characteristic of P450 enzymes. Comparison of deduced amino acid sequence with other CYP members shows that the CYP319A1 is more closely related to CYP4 family, but its overall identity to the CYP4 family is less than 40%. Therefore, it was assigned to a new P450 family by the P450 nomenclature committee. A pseudogene which shares high homology with the CYP319A1 was identified. Analysis of genomic sequence of the pseudogene indicated that the pseudogene contains two additional DNA inserts in the coding region, which disrupt the open reading frame. RT-PCR analysis showed that CYP319A1 is expressed in both susceptible and acaricide-resistant ticks.

Cloning and sequencing of a putative acetylcholinesterase cDNA from Boophilus microplus (Acari: Ixodidae).

Using a strategy based on degenerate primers derived from acetylcholinesterase (AChE) from other species, we cloned and sequenced a putative AChE cDNA from the southern cattle tick, Boophilus microplus (Canestrini). The sequence has a high degree of homology to sequences of AChE from other species reported in the GenBank. The open reading frame of 1,689 bp, corresponding to a deduced sequence of 563 amino acids, has conserved regions and features shared by the AChE family, necessary for its catalytic activity. No differences were found in the putative cDNA sequences from organophosphorus acaricide (OP) resistant and susceptible strains. The results suggest that this putative AChE gene is not involved in resistance to OP compounds as a mutated gene in the resistant strain studied. However, differences were detected, with a probe derived from this cDNA, in DNA fragments after digestion of genomic DNA from different strains with restriction nucleases. This indicates polymorphism in this gene in B. microplus.

Sequence analysis of the knockdown resistance-homologous region of the para-type sodium channel gene from pyrethroid-resistant Boophilus microplus (Acari: Ixodidae).

Using reverse transcription polymerase chain reaction and degenerate oligonucleotide primers, a partial para-homologous sodium channel cDNA was obtained from the southern cattle tick, Boophilus microplus (Canestrini). The cDNA sequence encoded the region in which knockdown resistance (kdr)-type mutations have been identified in numerous insect species. Comparison of deduced amino acids from the cDNA sequence showed high similarity with sodium channels from other species, particularly in highly conserved repeat domains of the sodium channel. Analysis of the kdr-homologous region of the genomic DNA sequences from several susceptible and pyrethroid-resistant tick strains did not detect mutations. The result suggests novel mutations in the sodium channel gene or metabolic detoxification may be involved in the resistance to pyrethroids in this tick.

Identification of a point mutation in the para-type sodium channel gene from a pyrethroid-resistant cattle tick.

To investigate the molecular mechanism of resistance to pyrethroids in the southern cattle tick, Boophilus microplus, we have obtained and sequenced a partial para-homologous sodium channel cDNA from susceptible and pyrethroid-resistant tick strains. A point mutation that results in an amino acid change from Phe to Ile was identified in the highly conserved domain IIIS6 of the homologous sodium channel from ticks that are highly resistant to pyrethroid acaricides. This mutation is at a location different from those reported in the same gene in pyrethroid-resistant insects.

Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae).

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.

Comparison of genomic DNA in various strains of Boophilus microplus (Acari: Ixodidae).

The conventional method of identifying acaricide resistance in a suspect tick population by the United Nations Food and Agriculture Organization packet assay is a laborious and time-consuming process. DNA probes have been demonstrated as rapid and accurate tools for detecting pesticide resistance in insect species. Random-amplified polymorphic DNA (RAPD) has been used by other groups to differentiate species of mosquitoes and populations within a mosquito species. By using different arbitrary oligonucleotides as primers with RAPD, we have demonstrated that various strains of Boophilus microplus (Canestrini) show different patterns of DNA fragments on agarose gel electrophoresis. The unique DNA fragments may be useful for developing probes that can detect acaricide resistance in field pest populations.

Lack of dietary calcium effect on chlordecone increased white blood cell count, total iron, and iron-binding capacity in serum of rat.

Male Sprague-Dawley rats were treated with 0, 1, 10, 50, and 100 ppm of chlordecone (Cd) mixed in calcium-sufficient (Ca-S) or calcium-deficient (Ca-D) diet for 15 days. The control rats fed with Ca-D diet exhibited a significant increase in white blood cell (WBC) counts compared to the rats fed with Ca-S diet. Dietary calcium (Ca), however, did not elicit any significant effect on total iron content and iron-binding capacity (transferrin) of control rats, whereas Cd at higher concentrations significantly increased WBC counts, total iron, and iron-binding capacity in serum of both Ca-S and Ca-D rats. The data suggest that dietary Ca did not alter Cd-increased WBC count, total iron, and iron-binding capacity in serum of rat.

Isopimpinellin is not phototoxic in a chick skin assay.

Synthetic isopimpinellin (5,8-dimethoxypsoralen), confirmed to contain as impurities only trace quantities at most of psoralen, bergapten (5-methoxypsoralen) and xanthotoxin (8-methoxypsoralen), is not phototoxic when tested in a chick skin bioassay system. These findings are at variance with earlier studies showing isopimpinellin to be phototoxic against chick skin and support the conclusion that isopimpinellin is photobiologically inactive. As recently proposed by others, the several reports of isopimpinellin photoactivity are most likely attributable to contamination by small amounts of highly active psoralens such as bergapten or xanthotoxin.

Influence of dietary calcium on chlordecone-induced biochemical changes in serum of rat.

Male, Sprague-Dawley rats were treated with different (1, 10, 50, and 100 ppm) concentrations of chlordecone (Cd) in calcium-sufficient (Ca-S) or calcium-deficient (Ca-D) diet for 15 days. No significant changes in serum total proteins were observed. However, serum nonprotein nitrogen compounds (urea, uric acid, and creatinine) and glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, creatine kinase, and alkaline phosphatase were significantly increased at 50 and 100 ppm of Cd. Chlordecone induced more increase in these serum components of rats fed with Ca-D as compared to Ca-S diet. Increased serum nonprotein nitrogen compounds and enzymes indicate Cd-altered glomerular and hepatic functions.

Metabolism of [14C]propionic acid in broiler chicks.

Broiler chicks were given 4 or 120 mumol propionic[1-14C] acid by gavage to determine its chemical fate and distribution of radiolabel among organs and tissues [foregut (crop, gizzard, and proventriculus), intestine (small and large), ceca, liver, and serum]. At 15 and 60 min postgavage, most of the extractable radiolabel remaining in the chicks was found in the foregut. Significantly higher percentages of the administered radiolabel were detected at 15 min in the serum and liver extracts of chicks given 120 mumol of propionic acid than in chicks given only 4 mumol. After 15 min, 41 and 30%, respectively, of the total radiolabel administered was accounted for in extracts of the digestive tract and tissues of chicks given 4 or 120 mumol of [14C]propionic acid. Only about 12% of the administered radiolabel was extracted from body compartments at 60 min postgavage from chicks given 4 mumol of propionic acid. Collection of respired [14C]CO2 during a 3-h postgavage period indicated that orally administered propionic acid is largely (about 75%) used as an energy source or is metabolized and assimilated into body components. The present studies indicate that little if any dietary propionic acid reaches the lower digestive tract and the ceca.

The toxic effects of mature flatpea (Lathyrus sylvestris L cv Lathco) on sheep.

The toxic effects of mature, seed-bearing flatpea (Lathyrus sylvestris L cv Lathco) hay on sheep was studied in a feeding trial with 25 adult ewes. Five ewes were barren; 20 were in the last 10 w of pregnancy. The ewes were blocked by weight and reproductive status for assignment to treatment groups. Four groups of 5 ewes each were allotted to 1 of the following diets: 0% (controls), 35.0%, 52.5% or 70.0% flatpea (FP); and 1 group of 5 ewes was allotted to a diet of increasing levels of 17.5, 35.0, 52.5 and 70.0% FP. Flatpea hay was harvested in the mature, seed-bearing stage, pelleted, reground and incorporated into a basal (control) diet of 30% corn + 70% alfalfa by substitution of an appropriate amount of FP for alfalfa. The resulting diets were fed for up to 42 days. The toxic nature of FP was expressed by the following dose-related responses: Fluctuating reductions in feed intake upon initiation of diets containing 35% FP and above; occurrence of a potentially fatal, acute central nervous system syndrome at FP levels as low as 35%; and reduced weight gain at FP levels of 52.5% and above. A wasting condition was observed in 1 ewe fed 52.5% FP. Four of 5 ewes and 1 lamb fed increasing levels of FP developed acute poisoning. One of these ewes developed acute neurotoxicosis when consuming 52.5% FP; 3 other ewes and 1 40-d-old orphaned lamb developed neurotoxicosis upon raising the FP level to 70.0%.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of dietary calcium and chlordecone on cholinesterase, triglycerides, low density lipoproteins, and cholesterol in serum of rat.

Male, Sprague-Dawley rats were treated with 0, 1, 10, 50, 100 ppm chlordecone (Cd) mixed in calcium-sufficient (Ca-S) or calcium-deficient (Ca-D) diet for 15 days. A significant decrease in body weight gain was observed in 100 ppm of Cd-treated rats. Cholinesterase (ChE) activity was significantly decreased in serum of Ca-D rats. Chlordecone did not alter serum ChE activity in both Ca-S and Ca-D rats. However, Cd decreases serum triglycerides, low density lipoproteins (LDL) and cholesterol in both Ca-S and Ca-D rats. Rats fed with Ca-S or Ca-D diet exhibited differential sensitivity to Cd-toxicity. Decreased levels of serum triglycerides, LDL and cholesterol suggest that Cd might interfere in lipid metabolism.

Fate of [(14)C]xanthotoxin (8-methoxypsoralen) in laying hens and a lactating goat.

The metabolism of xanthotoxin, a naturally occurring furanocoumarin photosensitizer, was studied in laying hens and a lactating goat treated with single oral doses equivalent to 10 mg xanthotoxin/kg of body weight. Within 48 h, essentially all of the administered radiocarbon was eliminated in the excreta of the laying hens, while in the goat 92% and 3% were excreted in the urine and feces, respectively. Radiocarbon residues in the milk, egg white, and egg yolk were low. Xanthotoxin, 8-hydroxypsoralen (xanthotoxol), 6-(7-hydroxy-8-methoxycoumaryl)-acetic acid (HCA) and 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyacetic acid (HCHA) were identified in the excreta of laying hens. In the goat, xanthotoxin was metabolized to HCA, HCHA, xanthotoxol, 5,8-dihydroxypsoralen, psoralenquinone, 5-hydroxy-8-methoxy-psoralen and 3[5-(6-hydroxy-7-methoxybenzofuryl)]-propanoic acid. Thus, identified metabolites in one or both of these species arose throughO-demethylation, oxidative cleavage of the furan ring, hydroxylation, reduction, oxidation, and hydrolysis of the lactone ring.

The individual and combined toxicity of kojic acid and aflatoxin in broiler chickens.

The individual and combined effects of kojic acid and aflatoxin were studied in male broiler chicks (Peterson x Hubbard). The experiment had a two by two factorial arrangement of treatments with dietary treatments of 0 and 2,500 mg kojic acid/kg feed and 0 and 2.5 mg aflatoxin/kg feed. The broilers were obtained at 1 day of age and housed in electrically heated batteries with feed and water available for ad libitum intake until they reached 3 wk of age. The toxicity of kojic acid was characterized by significant (P less than .05) reductions in body weight, the relative weight of the bursa of Fabricius, serum cholesterol concentration, and serum alkaline phosphatase activity, and by significant (P less than .05) increases in the relative weight of the pancreas, proventriculus, and gizzard, and serum concentrations of uric acid and triglycerides. Aflatoxicosis was characterized by significant (P less than .05) reductions in body weight, serum concentrations of total protein, albumin, cholesterol, and inorganic phosphorus, serum glutamic oxalacetic transaminase activity, and mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. Significant (P less than .05) increases in the relative weight of the liver, kidney, spleen, pancreas, proventriculus, and heart, and the serum pyruvic transaminase activity were also caused by aflatoxin alone. The only significant (P less than .05) interaction between kojic acid and aflatoxin, which can best be described as antagonistic, was seen through an increase in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. These data indicate that kojic acid is not an aflatoxin synergist at the levels used in the present study.

Comparative fate of the tritiated trichothecene mycotoxin, T-2 toxin, in chickens and ducks.

A tritiated preparation of the trichothecene mycotoxin, T-2 toxin, was administered as a single oral dose to 21-day-old male broiler (Hubbard x Hubbard) chickens and White Pekin ducks. There were few significant differences between the two species in metabolism, tissue retention, and excretion of T-2 toxin and its metabolites. On the basis of the data obtained, the differences in toxicological sensitivity to T-2 toxin known to exist between these two species cannot likely be attributed to differences in the metabolism or elimination of T-2 toxin from the body.

Toxic effects of kojic acid in the diet of male broilers.

The toxicological effects induced by the administration of kojic acid were characterized in young male broiler chickens (Hubbard x Peterson). The experimental design consisted of six dietary treatments of kojic acid (0, .5, 1, 2, 4, and 8 g/kg feed) and four replicates of 10 broilers per replicate. Kojic acid significantly (P less than .05) decreased growth rates at concentrations greater than 2 g basic acid/kg feed. Kojic acid in higher concentrations also significantly increased the relative weights of the proventriculus, gizzard, pancreas, and liver, and significantly decreased the relative weight of the bursa of Fabricius at necropsy (Day 21). Hematological changes included a significant increase in the number of red blood cells, a significant increase in packed-cell volume, and a significant decrease in mean corpuscular volume. Also occurring was a significant increase in the serum concentrations of total protein, albumin, cholesterol, and triglycerides. Hepatotoxicity of dietary kojic acid was evident through a significant increase in the activities of serum glutamic oxalacetic transaminase and creatine kinase and a significant decrease in the activity of alkaline phosphatase. A significant increase in serum uric acid concentration was indicative of nephrotoxicity, and a significant increase in serum glucose concentration was indicative of pancreatic toxicity. A significant decrease in colonic temperature was also observed. Because the toxic effects of kojic acid were only observed at concentrations greater than 2 g kojic acid/kg feed, this mycotoxin alone does not appear to pose a serious economic threat to the poultry industry.

Induction of glutathione S-transferases in genetically inbred male mice by dietary ethoxyquin hydrochloride.

1. Constitutive and ethoxyquin hydrochloride (EQ-HCl)-induced hepatic glutathione (GSH) S-transferase, GSH reductase, and GSH peroxidase activities were determined in 5 strains of 8-10 week old inbred male mice. 2. The constitutive GSH S-transferase (GST) activity varied from 2.9 (SJL/JCR) to 8.9 (C57BL/6NCR) mumol product formed/min/mg protein and the corresponding values for the EQ-HCl-treated mice were in the range of 15.3-25.3 mumol product formed/min/mg protein. 3. EQ-HCl induced GST activity in all the strains examined and this contrasted to the induction activity of Aroclor 1254 which was strain-dependent. GST activity was induced 2.9-fold in Aroclor 1254-responsive (C57BL/6) and 2.8-fold in non-responsive (DBA/2) mice, respectively.

Fate of [14C]coumaphos after dermal application to lactating goats as a pour-on formulation.

Two lactating Nubian goats were dermally treated with [14C]coumaphos (O-[3-chloro-4-methyl-2-oxo-2H-benzopyran-7-yl] O,O-diethyl phosphorothioate) as a 4% active ingredient pour-on formulation. Doses were administered, along the dorsal midline from withers to sacrum, at a rate equivalent to 14 mg of coumaphos/kg of body weight. During the 7 days after treatment, an average of less than 0.1, 4.7, and 1% of the administered dose was eliminated in the milk, urine, and feces, respectively. When goats were killed after 7 days, about 45% of the administered radiocarbon remained on the hair and skin, and this consisted almost entirely of intact coumaphos. Residues in selected tissues collected after 7 days were, in every case, less than 1 mg of coumaphos equivalent/kg of tissue, with highest residues in adipose tissue, followed by residues in kidney and liver. In milk, residue amounts plateaued after about 2 days and remained relatively constant at about 0.1 mg of coumaphos equivalent/kg of milk. Residues in adipose tissue and in milk consisted mainly of unmetabolized coumaphos. In urine, most radiocarbon was present as metabolites of coumaphos, but in feces, most radiocarbon was present as the intact parent compound. Coumaphos was absorbed slowly and at a constant rate after dermal application to lactating goats as a pour-on formulation.

Constitutive and Aroclor 1254-induced hepatic glutathione S-transferase, peroxidase and reductase activities in genetically inbred mice.

1. Constitutive and Aroclor 1254-induced hepatic glutathione (GSH) S-transferases, GSH peroxidase and GSH reductase activities were determined in 12 strains of 8-10 week-old inbred male mice. 2. The constitutive GSH S-transferase activity varied from 2.5 (SJL/JCR) to 8.9 (C57BL/6N) mumol/min/mg protein and the corresponding values for the Aroclor 1254-treated mice were in the range of 7.1-23.0 mumol/min/mg protein. Aroclor 1254 significantly induced GSH S-transferase activity in all mice, however, significant interstrain differences were found in inducibility. 3. Aroclor 1254-treatment caused a 4.2-fold induction of GSH S-transferase in NFS/NCR but only a 1.4-fold increase in AKR/NCR mice. Aroclor 1254 significantly induced GSH reductase in all strains studied while GSH peroxidase activity decreased in these mice. 4. The range of hepatic GSH levels in control and Aroclor 1254-treated mice was relatively narrow for both groups (6.59-11.25 microM/g wet tissue).

Inhibition of macrophage adenylate cyclase by the alpha-methylene-gamma-lactone moiety of sesquiterpene lactones from forage plants.

Inhibition of murine macrophage adenylate cyclase activity by sesquiterpene lactones isolated from toxic forage plants was highly correlated with the presence of the alpha-methylene-gamma-lactone moiety on the molecule (ie, hymenovin and helenalin). Tenulin, a sesquiterpene lactone which does not contain this reactive moiety, caused minimal inhibition of the enzyme. Reaction of the alpha-methylene-gamma-lactone moiety of hymenovin and helenalin with cysteine decreased the number of reactive moieties available to alkylate the enzyme, thus decreasing the inhibition of adenylate cyclase by these 2 sesquiterpene lactones. As the reaction time available for the reduction by cysteine of the alpha-methylene-gamma-lactone moiety decreases, the amount of adenylate cyclase inhibition increases. Stimulation of the hymenovin- or helenalin-inhibited adenylate cyclase by prostaglandin E1 or E2 or by sodium fluoride did not reverse the inhibition of the enzyme, but did stimulate the undamaged adenylate cyclase in the sesquiterpene lactone treatment groups to the same degree as in the nontreated control. These data indicate that sesquiterpene lactones containing an alpha-methylene-gamma-lactone moiety are potent inhibitors of macrophage adenylate cyclase activity. This moiety may have a significant role in the toxicity of some sesquiterpene lactones in poisonous plants when ingested by livestock.