The effect of selected in ovo green light photostimulation periods on post-hatch broiler growth and somatotropic axis activity.

Targeted in ovo green light (GL) photostimulation during the last days of broiler egg incubation increases embryonic expression of the somatotropic axis, similar to in ovo green light photostimulation from embryonic day (ED) 0 to the end of incubation. The aim of this study was to examine the effect of selected in ovo GL photostimulation periods on post-hatch broiler growth. Four hundred twenty fertile broiler eggs were divided into 7 treatment groups: the first incubated in the dark (standard conditions) as a negative control; the second incubated under monochromatic GL from ED0-ED20 (positive control); the third group incubated under monochromatic GL light from ED15-ED20; the fourth, fifth and sixth groups were incubated under monochromatic GL on ED16, ED17, and ED18, respectively; and the seventh group was incubated under monochromatic GL from ED18-ED20. All illumination was provided intermittently using LED lamps. After hatch, all chicks were transferred to a controlled room under standard rearing conditions. The group incubated under green light from ED18 until hatch showed similar results to the positive control group in body weights, as well as breast muscle weights (as % of body weights), and an elevation in the somatotropic axis activity during the experiment. We suggest that broiler embryos can be exposed to in ovo GL photostimulation from ED18 until hatch (hatching period), and still exhibit the same performance as obtained by photostimulation from d 0 of incubation.

In ovo green light photostimulation during the late incubation stage affects somatotropic axis activity.

Targeted green light photostimulation during the last stage of broiler incubation increases expression of the somatotropic axis. The purpose of this study was to further shorten the in ovo green light photostimulation and determine the critical age for photostimulation in broilers embryos, as a future strategy for broiler incubation. Fertile broilers eggs (n = 420) were divided into 5 treatment groups. The first group was incubated under standard conditions (in the dark) as the negative control group. The second was incubated under intermittent monochromatic green light using light-emitting diode lamps with an intensity of 0.1 W/m2 at shell level from embryonic day (ED) 0 of incubation until hatch, as a positive control. The third, fourth, and fifth groups were incubated under intermittent monochromatic green light from ED 15, 16, and 18 of incubation, respectively, until hatch. All treatment groups showed elevated somatotropic axis expression compared with the negative control, with the group incubated under monochromatic green light from ED 18 until hatch showing results closest to the positive control. This suggests that broiler embryos can be exposed to in ovo green light photostimulation from a late stage of incubation (when transferring the eggs to the hatchery) and exhibit essentially the same outcome as obtained by photostimulation during the entire incubation period.

In-ovo green light photostimulation during different embryonic stages affect somatotropic axis.

Previous studies demonstrated that in-ovo photostimulation with monochromatic green light increased the somatotropic axis expression in broilers embryos. The objective of the current study was to detect the critical period for in-ovo GL photostimulation, in order to find the optimal targeted photostimulation period during the incubation process. Three hundred thirty-six fertile broiler eggs were divided into 4 groups. The first group was incubated under dark conditions as a negative control. The second incubated under intermittent monochromatic green light using light-emitting diode (LED) lamps with an intensity of 0.1 W\m2 at shell level from d 0 of the incubation as a positive control. The third group incubated under intermittent monochromatic green light from d 10 of the incubation. The last group incubated under intermittent monochromatic green light from d 15 of the incubation. In-ovo green light photostimulation from embryonic d 0 (ED0) increased plasma growth hormone (GH), as well as hypothalamic growth hormone releasing hormone (GHRH) and liver growth hormone receptor (GHR) and insulin-like growth factor-1 (IGF-1) mRNA levels. In-ovo green light photostimulation from ED10 increased the GH plasma levels compared to the negative control group, without affecting somatotropic axis mRNA genes expressions of GHRH, GHR, and IGF-1. In-ovo green light photostimulation from ED15 caused an increase in both the plasma GH levels and the somatotropic axis mRNA genes expressions of GHRH, GHR, and IGF-1, compared to the negative control group. These results suggest that the critical period of somatotropic axis acceleration by GL photostimulation start at 15 d of incubation.

In-ovo monochromatic green light photostimulation enhances embryonic somatotropic axis activity.

Previous studies demonstrated that in ovo photostimulation with monochromatic green light increases body weight and accelerates muscle development in broilers. The mechanism in which in ovo photostimulation accelerates growth and muscle development is not clearly understood. The objective of the current study was to define development of the somatotropic axis in the broiler embryo associated with in ovo green light photostimulation. Two-hundred-forty fertile broiler eggs were divided into 2 groups. The first group was incubated under intermittent monochromatic green light using light-emitting diode (LED) lamps with an intensity of 0.1 W\m2 at shell level, and the second group was incubated under dark conditions and served as control. In ovo green light photostimulation increased plasma growth hormone (GH) and prolactin (PRL) levels, as well as hypothalamic growth hormone releasing hormone (GHRH), liver growth hormone receptor (GHR), and insulin-like growth factor-1 (IGF-1) mRNA levels. The in ovo photostimulation did not, however, increase embryo's body weight, breast muscle weight, or liver weight. The results of this study suggest that stimulation with monochromatic green light during incubation increases somatotropic axis expression, as well as plasma prolactin levels, during embryonic development.

Role of the serotonergic axis in the reproductive failure associated with aging broiler breeder roosters.

Reproductive failure associated with aging is a well-known phenomenon. However, the mechanism by which this failure occurs in broiler breeder roosters is still unclear. A previous study conducted in our laboratory, comparing young and aging broiler breeder roosters, demonstrated an elevation in hypothalamic vasoactive intestinal peptide (VIP) and pituitary prolactin (PRL) gene expression accompanied by a deterioration of gonadal axis function. This resulted in a decrease in semen-quality variables as roosters aged. The objective of this study was to examine the involvement of the serotonergic axis in the age-associated reproductive failure in broiler breeder roosters. Cobb roosters aged 64 wk were divided into 3 groups (n = 20 each): parachlorophenylalanine (PCPA) administration, active immunization against chicken VIP, and controls. At 69 wk of age, each group was divided into 2 equal subgroups: 1 received ovine PRL and the other served as controls. Weekly semen volume, concentration and motility, and plasma testosterone, estradiol, and PRL concentrations were examined. At the end of the experiment, roosters were euthanized, testes were weighed, and hypothalamus and pituitary were removed to assay the expression of genes encoding hypothalamic GnRH-I, pituitary FSH, pituitary LH, hypothalamic VIP, and pituitary PRL. Both PCPA administration and active immunization against chicken VIP significantly increased testis weight, semen volume, sperm concentration, ejaculation grade, plasma testosterone level, and GnRH-I, FSH and LH gene expression compared with controls (P ≤ 0.05). In addition, a decrease in plasma estradiol and PRL concentrations and VIP and PRL gene expression was observed in PCPA- and VIP-immunized birds compared with controls (P ≤ 0.05). Administration of PRL in all groups decreased gonadal axis function and semen-quality variables (P ≤ 0.05). Collectively, these results suggest that the increasing expression levels of the serotonergic axis in aging broiler breeder roosters inhibit proper gonadal function and reproductive performance. This article establishes for the first time the inhibitory role of serotonin on reproduction in aging roosters.

Age-related changes in gonadal and serotonergic axes of broiler breeder roosters.

Fertility of domestic roosters decreases at ≈ 50 wk of age. In a previous study on aging white leghorn roosters, low fertility was accompanied by low levels of both hypothalamic vasoactive intestinal peptide (VIP) and pituitary prolactin (PRL) mRNA expression; however, their role in aging broiler breeder rooster reproduction is still unclear. In this study we compared reproductive activities of young (35-wk-old) and aging (73-wk-old) broiler breeder roosters. Weekly semen volume; concentration and ejaculation grade; and concentrations of plasma testosterone, estradiol, and PRL were examined. Every other week, 10 roosters from each group were euthanized, their testes weighed, and hypothalamus and pituitary removed to determine mRNA expression of hypothalamic GnRH-I, pituitary FSH, pituitary LH, hypothalamic VIP, and pituitary PRL. Aging roosters had significantly lower testis weight and semen volume, sperm concentration, ejaculation grade and plasma testosterone and low hypothalamic GnRH-I, pituitary FSH, and pituitary LH mRNA expression than young roosters (P ≤ 0.05). Aging roosters had higher concentrations of plasma estradiol and PRL and higher hypothalamic VIP and pituitary PRL mRNA expression than young roosters (P ≤ 0.05). We suggest that PRL, which is known to inhibit the gonadal axis, and its releasing factor, VIP, play an important role in the reproductive failure associated with age in broiler breeder roosters.

The effect of active immunization against vasoactive intestinal peptide (VIP) and inhibin on reproductive performance of aging White Leghorn roosters.

Decreasing fertility in aging domestic roosters is a well-known phenomenon. Aging is manifested by a decrease in plasma testosterone level, testis function, and spermatogenesis, resulting in a low level of fertility. The roles of vasoactive intestinal peptide (VIP) and testicular inhibin in this aging process are not clear. The effects of active immunization against VIP, inhibin, or the combination of both hormones on the reproduction of aging White Leghorn (WL) roosters were assayed. In experiment 1a, 60 White Leghorn roosters (67 wk of age) were divided into 4 groups (n = 15/group). The first group was actively immunized against VIP, the second against inhibin, the third against VIP and inhibin, and the fourth served as a control. Active immunization against VIP decreased semen quality parameters, plasma steroid levels, and gene expression of gonadotropin-releasing hormone-I (GnRH-I), follicle-stimulating hormone (FSH), luteinizing hormone (LH), LH receptor, VIP, and prolactin (Prl). Immunization against inhibin increased some of the semen quality parameters and FSH mRNA gene expression but decreased inhibin gene expression. In experiment 1b, at 94 wk of age, we took the actively immunized against VIP group and the control group and divided them into 2 subgroups (n = 7 or 8): the first group was injected with 1 mg of ovine Prl (oPrl) daily for 7 d, and the second group served as a control. Administration of oPrl to previously VIP-immunized birds significantly elevated semen quality parameters. We suggest that VIP, Prl, and inhibin have an important effect on the reproductive axis in aging roosters. Active immunization against VIP-depressed reproductive activity and Prl administration restored their reproduction, indicating that both VIP and Prl are essential for reproduction in aging roosters. Immunization against inhibin improved FSH mRNA gene expression, suggesting a negative role of inhibin on FSH secretion in aging roosters. Not all semen quality parameters increased significantly after immunization against inhibin, even though FSH mRNA gene expression increased, suggesting interference in testicular function in aging roosters.

The effect of active immunization against vasoactive intestinal peptide and inhibin on reproductive performance of young White Leghorn roosters.

The neuropeptides vasoactive intestinal peptide (VIP) and gonadal inhibin have long been considered putative regulators of reproduction in hens. However, their role in young roosters remains unclear. We studied the effect of active immunization against VIP, inhibin, and a combination of both hormones on reproduction in young White Leghorn roosters. At 13 wk of age, White Leghorn roosters (n = 60) were split into 4 groups (n = 15). One group was actively immunized against VIP, the second against inhibin, the third against both VIP and inhibin, and the fourth, untreated, served as a control. Active immunization against VIP enhanced reproductive parameters as manifested by increased semen quality, plasma steroid levels, and mRNA gene expression of hypothalamic gonadotropin-releasing hormone-I, pituitary follicle-stimulating hormone, pituitary luteinizing hormone (LH), and decreased mRNA gene expression of hypothalamic VIP, pituitary prolactin, and testicular LH receptor. In contrast, immunization against inhibin decreased reproductive parameters such as semen quality, plasma steroid levels, mRNA gene expression of pituitary follicle-stimulating hormone and testicular inhibin. The combined treatment showed the greatest increase in semen quality parameters, plasma steroid levels, and mRNA gene expression of hypothalamic gonadotropin-releasing hormone-I, pituitary follicle-stimulating hormone, pituitary LH, and testicular LH receptor. Moreover, it significantly reduced mRNA gene expression of hypothalamic VIP and pituitary prolactin and mildly reduced that of testicular inhibin. These results suggest that VIP plays a negative role, at a young age, in reproduction of roosters that is similar to that in hens and that inhibin is as important in reproductive function in young roosters as in mammals.

The role of retinal and extra-retinal photostimulation in reproductive activity in broiler breeder hens.

Photostimulation of retinal photoreceptors, which are sensitive to green light, appears to inhibit reproductive activity in birds, whereas photostimulation of extra-retinal photoreceptors, which are sensitive to red light, accelerates it. The objective of this study was to determine the effect of either retinal or extra-retinal photostimulation on reproductive activities of broiler breeder hens. At 23 wk of age, Cobb hens (N=135) were divided into 9 rooms with individual cages (n=15). At 24 wk of age, 3 rooms were photostimulated (14L:10D) with white light (Control, n=45). Six rooms had 2 parallel lighting systems, red (660 nm) and green (560 nm), which were both on during 6 out of 14 h of the light period. Then, in 3 of these rooms, the green light was turned off and hens were exposed to a total of 14 h of red light (Red, n=45), and in the other 3, the red light was turned off and green lighting continued for a total of 14 h (Green, n=45). The Green group had reduced egg production; reduced plasma concentrations of ovarian steroids; reduced luteinizing hormone (LH)-beta, vasoactive intestinal peptide (VIP), and prolactin mRNA expression; and greater retinal green opsin mRNA expression (P < or = 0.05). The Red group had greater egg production; greater gonadotropin-releasing hormone-I (GnRH-I) and red opsin gene expression in the hypothalamus; and lesser green opsin gene expression in the retina (P < or = 0.05). We suggest that selective photostimulation of extra-retinal photostimulation as opposed to retinal photostimulation is a key factor in the determination of successful reproduction of broiler breeder hens.

Corticosterone oxidative neutralization by 11-beta hydroxysteroid dehydrogenases in kidney and colon of the domestic fowl.

In mammalian organs involved in sodium reabsorption, the 11-beta hydroxysteroid dehydrogenases (11betaHSDs) oxidize glucocorticoids (GC) from their 11-alcohol form to their 11-keto state and therefore prevent their binding to mineralocorticoid (MC) receptors (MR) and the development of a MC excess syndrome. In birds the information about 11betaHSDs and GC metabolism in such organs is scarce. Herein, we report the expression and enzymatic activity of 11betaHSDs in the kidney and colon of chickens. Both organs express 11betaHSD2-like mRNA. With NAD(+), microsomes from both tissues oxidized corticosterone (CS) into 11-dehydrocorticosterone (DHC) with K(m) of 200 and 20nM and V(max) of 13 and 2pmol/mg protein/min in the kidney and colon, respectively. Thiram, a specific 11betaHSD2 inhibitor, suppressed this oxidation in kidney. The expression and action of the putative 11betaHSD3 were also tested. The chicken colon, and to a greater extent the kidney, expressed 11betaHSD3-like mRNA. Microsomal fractions from both tissues oxidized CS into DHC in the presence of NADP(+) with K(m) of 150 and 4nM and V(max) of 5 and 0.3pmol/mg protein/min for the kidney and the colon, respectively. This oxidation was not affected when NADP(+) conversion into NAD(+) was inhibited by excess pyrophosphate or a phosphatase inhibitor cocktail. In microsomes of chicken's duodenum, where 11betaHSD1-like mRNA expression is high, NADP(+)-dependent oxidation of CS into DHC has a low-affinity K(m) of 1130nM. This study documented the expression and activity of two enzymes that convert CS into DHC, one is 11betaHSD2-like and the other is similar to the putative mammalian 11betaHSD3.

Distinct features of dehydrocorticosterone reduction into corticosterone in the liver and duodenum of the domestic fowl (Gallus gallus domesticus).

The mammalian 11-beta hydroxysteroid dehydrogenase type 1 (11 betaHSD1) reduces glucocorticoids (GC) at C11 from the 11-keto-GC nonactive form to the 11-hydroxy-GC active form, an action essential for survival. Whereas GC metabolism at C11 and the role of 11 betaHSD1 are studied extensively in mammals, information about these in birds is scattered. Herein, we report the GC bidirectional metabolism in chickens. In hens' liver and duodenal mucosa, 11 betaHSD1-like mRNA expression was detected; and 11 betaHSD1-like immunoreactivity was found linked to membranes of hepatocytes and duodenal enterocytes. With either NADH or NADPH, the membranal fraction of liver and duodenal mucosa converted dehydrocorticosterone (A) into corticosterone (B) with K(m) (1.1-8.7 microM) and V(max) (10-40 pmol/mg protein/min) values similar to those reported for mammalian 11 betaHSD1. In the presence of NADP(+) or NAD(+), these membranal fractions oxidized B into A. With either NADPH or NADH, the cytosol of chicken liver and duodenal mucosa reduced A into B (K(m) of 1.1 - 2.3 microM and V(max) of 260-960 pmol/mg protein/min). These cytosolic fractions did not convert any amount of B into A when incubated with either NADP(+) or NAD(+). This may suggest that chicken liver and duodenal mucosa express 11 betaHSD1 that is a membrane-bound oxoreductase which uses both NADPH/NADP(+) and NADH/NAD(+) as cosubstrates. The substantial reduction of A into B (but no conversion of B into A) found in the cytosol is most likely executed by a unidirectional soluble reductase, different than 11 betaHSD1.

Effect of hypothalamic electrolytic lesions in White Leghorn and broiler male cockerels.

1. This study compared the effect of bilateral electrolytic lesions of the basomedial hypothalamus (HL) in broiler and White Leghorn (WL) males. 2. Hypothalamic lesions were placed in WL at 10 weeks of age (body weight 1.1 kg) and in broilers, either at 6 weeks (body weight 1.5kg) or at 10 weeks of age (body weight 3.4kg). They were fed ad libitum until autopsy at 16 and 17 weeks of age for broilers and WL, respectively. 3. Hypothalamic lesions caused obesity (high percentage weight of abdominal adipose tissue) in both strains. Obese fowls with unimpaired reproductive systems were classified as OB and those with functional castration as FC (functionally castrated) or FCLC (functionally castrated with large comb). 4. All post-HL syndromes-OB, FC and FCLC-were present in WL, whereas all obese broilers (which are immature at this age) were classified as OB. 5. The percentage weight of abdominal adipose tissue in OB broilers was lower than in OB WL (3% vs 5%, respectively). 6. Daily food intake of OB broilers was higher than control at 12 to 15 weeks of age, regardless of time of placement of HL, whereas daily food intake of OB WL was significantly higher than that of control WL only during the first 2 weeks following HL. 7. Body weight of OB broilers at autopsy was 20% higher than control broilers, whereas body weight of OB WL was not significantly affected. 8. An additional group of broilers was reared to sexual maturity under food restriction until 28 weeks of age. HL were placed at 10 weeks of age (body weight 1.7 kg). Autopsy was performed after a 4-week period of ad libitum feeding. 9. There were OB as well as FC and FCLC among the HL, food-restricted broilers. Percentage weight of testes and spleen were reduced in OB fowls of both strains, but more so in OB WL. 10. Hyperphagia and weight gain were not observed during the ad libitum feeding period of those obese broilers after HL, indicating that hyperphagia and weight gain are secondary to obesity.

The role of prolactin in reproductive failure associated with heat stress in the domestic turkey.

Reproductive failure associated with heat stress is a well-known phenomenon in avian species. Increased prolactin (PRL) levels in response to heat stress have been suggested as a mechanism involved in this reproductive malfunction. To test this hypothesis, laying female turkeys were subjected to 40 degrees C for 12 h during the photo-phase daily or maintained at 24-26 degrees C. Birds in each group received oral treatment with parachlorophenyalanine (PCPA; 50 mg/kg BW/day for 3 days), an inhibitor of serotonin (5-HT) biosynthesis, or immunized against vasoactive intestinal peptide (VIP). Both treatments are known to reduce circulating PRL levels. Nontreated birds were included as controls. In the control group, high ambient temperature terminated egg laying, induced ovarian regression, reduced plasma luteinizing hormone (LH) and ovarian steroids (progesterone, testosterone, estradiol) levels, and increased plasma PRL levels and the incidence of incubation behavior. Pretreatment with PCPA reduced (P < 0.05) heat stress-induced decline in egg production, increase in PRL levels, and expression of incubation behavior. Plasma LH and ovarian steroid levels of heat stressed birds were restored to that of controls by PCPA treatment. As in PCPA-treated birds, VIP immunoneutralization of heat-stressed turkeys reduced (P < 0.05) circulating PRL levels and prevented the expression of incubation behavior. But it did not restore the decline in LH, ovarian steroids, and egg production (P > 0.05). The present findings indicate that the detrimental effect of high temperature on reproductive performance may not be related to the elevated PRL levels in heat-stressed birds but to mechanism(s) that involve 5-HT neurotransmission and the induction of hyperthermia.

Glucocorticoid administration during incubation: embryo mortality and posthatch growth in chickens.

The effects of glucocorticoids (GC) on embryonic mortality and posthatch BW were studied. Cortisol hemisuccinate or corticosterone in 0.1-mL vehicles were injected into the albumen of 7-d-old White Leghorn chicken embryos. Embryonic mortality rates and the age after injection at which death occurred were determined. When 0.02 to 20 microg cortisol per egg were injected in saline, total embryonic mortality rate increased in a doseresponse manner, with a median lethal dose (LD50) at 10 microg. Saline injection alone caused a similar mortality rate to that caused by injection of 2 microg cortisol (around 35%). However, whereas mortality among the cortisol-treated embryos was greatest on Days 16 to 18, most of the saline-treated embryos died around the time of injection. The lethal effect of corticosterone, which is endogenous GC in adult chickens, was compared to that of cortisol by injecting both in the same vehicle (a saline:ethanol mixture) and was found to be similar. However, when 2, 10, or 20 microg of corticosterone was injected in oil, mortality rates were lower than those caused by the matching doses of cortisol in saline, probably due to the lower diffusion rate of the steroid out of the oil carrier. Hatch weight was significantly lower in chicks treated with 10 and 20 microg cortisol, and BW of the latter was lower compared with control throughout the 3-mo observation. In conclusion, cortisol and corticosterone are equally active in causing embryonic mortality. Posthatch BW is affected only by GC doses that are equal to or greater than the LD50.

Short-term stress increases testosterone secretion from testes in male domestic fowl.

Prolonged stress inhibits the hypothalamus-pituitary-gonadal (HPG) axis and reduces plasma testosterone (T). However, enhanced secretion of luteinizing hormone (LH) and T has been documented during the initial stages of acute stress in mammals. This study assayed the effect of short-term stress on plasma T and corticosterone (B) in juvenile, pubertal, and adult White Leghorn cockerels. Stress was induced by brief physical restraint of caged juvenile (7 weeks), pubertal (17 weeks), and adult (40 weeks) cockerels, as well as 40-week-old adults reared together in a room lined with wood shavings (group reared). Blood was sampled immediately before restraint (0 time), at the end of a 10-min restraint period, and at 30, 60, and 180 min after 0 time. Restraint resulted in an initial increase in plasma T in all groups, along with a rise in B. Whereas B generally reached its peak level at the end of the restraining period, T peaked 20 min later. The maximum increase of T and B relative to prestress levels (T and B ratios) was similar in all groups, with median T ratio reaching 1.25-1. 5-about half that of the B ratio. Thus, the extent of T and B response to short-term stress was not influenced by basal levels of T, which were highest in adults, and basal levels of B, which were higher in caged adults than in group-reared adults. Injection of ACTH did not induce a greater increase in plasma T than in sham-injected controls. Further, the elevation of T in response to stress was extinguished in castrated adults, indicating that T is secreted from the testes rather than the adrenals in response to stress. When the same regime of blood sampling was applied to adults not subjected to restraint, the T ratio rose by up to 11 times. It can therefore be stipulated that T response depends on the type of stress applied, a factor that should be considered when investigating androgen levels in plasma.

Effect of phenethyl isothiocyanate on the metabolism of tobacco-specific nitrosamines by cultured rat oral tissue.

The effect of phenethyl isothiocyanate (PEITC) on the metabolism of N'-nitrosonornicotine (NNN) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cultured rat oral tissue was investigated. Two protocols were used. In one, oral tissue from untreated rats was cultured in the presence of 10 or 50 microM PEITC and either NNN or NNK. The levels of NNN and NNK metabolites released into the culture media were determined by HPLC analysis. The presence of 10 microM PEITC inhibited the formation of all NNN metabolites from 45 to 70% when the concentration of NNN was 1 microM or 10 microM. When the concentration of PEITC was 50 microM the extent of inhibition was from 70 to 90%. alpha-Hydroxylation of NNK was inhibited 70 to 90% and N-oxidation of NNK was inhibited 80 to 90% by 10 microM PEITC. Carbonyl reduction of NNK to NNAL was unaffected by 10 microM PEITC and only slightly inhibited by 50 microM PEITC. In the second protocol, rats were fed NIH-07 diet containing 3 mumol PEITC/g for 1-14 days. The metabolism of NNN by cultured oral tissue from these rats was decreased from 40 to 90% relative to that by tissue from control rats. NNK metabolism was inhibited 40 to 60%. The extent of inhibition was the same when rats were fed PEITC containing diet for 1 or 14 days. NNN and NNK are the only tobacco constituents which induce oral cavity cancer in an animal model. The results of this study suggest the possibility that PEITC may be useful as a chemopreventive agent for oral cavity cancer.

Effect of nicotine and tobacco-specific nitrosamines on the metabolism of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by rat oral tissue.

The effect of N'-nitrosoanatabine (NAT) and nicotine on the metabolism of N'-nitrosonornicotine (NNN) and 4-(methyl-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK) by cultured rat oral tissue was investigated. The effect of NNN on NNK metabolism and the effect of NNK on NNN metabolism was also determined. NNK inhibited NNN metabolism more than NNN inhibited NNK metabolism. NAT inhibited the metabolism of NNK but not of NNN. Nicotine, which is present at greater than 500 times the levels of NNN and NNK in smokeless tobacco, inhibited the metabolism of both nitrosamines. Inhibition of 1 microM NNN metabolism was greater than that of 1 microM NNK when the concentration of nicotine was 1, 10 or 100 microM. Nicotine at 100 microM inhibited the formation of all metabolites of NNN by 85-92%. These results suggest that NNN and nicotine may be metabolized by a common enzyme.

Comparative metabolism of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by cultured F344 rat oral tissue and esophagus.

The metabolism and DNA binding of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cultured F344 rat oral tissue and esophagus were investigated over a range of concentrations. The metabolites present in the culture media were separated by high performance liquid chromatography and were identified by comparison to standards. alpha-Hydroxylation of NNN, an esophageal carcinogen, was the major pathway for metabolism of this nitrosamine in both tissues. The metabolites formed from 2'-hydroxylation were between 3.0 and 3.9 times those formed from 5'-hydroxylation. 2'-Hydroxylation results in a pyridyloxobutylating species. DNA from esophagus cultured with [5-3H]NNN contained a pyridyloxobutylated adduct which upon acid hydrolysis released 3.8 pmol [5-3H]-4-hydroxy-1-(3-pyridyl)-1-butanone/mumol guanine. DNA from oral tissue cultured under the same conditions, where the extent of metabolism was the same, contained no measurable [5-3H]NNN DNA adduct. This suggests that factors, as yet unknown, cause the DNA of oral cavity tissue to be protected from pyridyloxobutylation by NNN. The metabolism of NNK by alpha-hydroxylation was as much as 10-fold less than the metabolism of NNN by this pathway in both tissues. alpha-Hydroxylation of NNK results in either a methylating species or a pyridyloxobutylating species. DNA from oral tissue cultured with [C3H3]NNK contained between 1.7 and 4.3 pmol 7-methylguanine/mumol guanine, respectively. No pyridyloxobutylated DNA (less than 0.2 pmol/mumol guanine) was detected in oral tissue incubated with [5-3H]NNK. The DNA from esophagi incubated with [C3H3]NNK contained no 7-methylguanine (less than 0.4 pmol/mumol guanine). The level of pyridyloxobutylation of DNA from esophagi incubated with [5-3H]NNK was 0.17 pmol/mumol guanine. The ability of the esophagus to metabolize NNN to a greater extent than NNK to a reactive species which pyridyloxobutylates DNA may be important in determining the carcinogenicity of NNN in the esophagus. In contrast, the metabolism of NNK to a methylating species by oral cavity tissue suggests that this tobacco-specific nitrosamine is important in tobacco-related oral cavity carcinogenesis.