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Made available in DSpace on 2013-09-27T14:54:12Z (GMT). No. of bitstreams: 1 WOS000288627700007.pdf: 187475 bytes, checksum: f44a03d706cf45e64484e91ab7b27272 (MD5) Previous issue date: 2011-02-01 Made available in DSpace on 2013-09-30T18:36:09Z (GMT). No. of bitstreams: 1 WOS000288627700007.pdf: 187475 bytes, checksum: f44a03d706cf45e64484e91ab7b27272 (MD5) Previous issue date: 2011-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:42:31Z No. of bitstreams: 1 WOS000288627700007.pdf: 187475 bytes, checksum: f44a03d706cf45e64484e91ab7b27272 (MD5) Made available in DSpace on 2014-05-20T13:42:31Z (GMT). No. of bitstreams: 1 WOS000288627700007.pdf: 187475 bytes, checksum: f44a03d706cf45e64484e91ab7b27272 (MD5) Previous issue date: 2011-02-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The objectives of this study were to determine the interval from ovulation to deviation and the diameter of the dominant (DF) and largest subordinate (SF) follicles at deviation in buffalo (Bubalus bubalis) heifers. Two methods of evaluation (observed vs. calculated) were used. FSH and LH profiles encompassing follicle deviation (Experiment 1), and the follicular diameter when the DF acquired ovulatory capacity (Experiment 2) were also determined. The time of deviation and the diameter of the of and the largest SF at deviation did not differ between observed and calculated methods. Overall, follicle deviation occurred 2.6 +/- 0.2 d (mean SEM) after ovulation, and the diameters of the DF and SF at deviation were 7.2 +/- 0.2 and 6.4 +/- 0.2 mm, respectively. No changes in plasma levels of FSH or LH were observed (P= 0.32 and P= 0.96, respectively). Experiment 2 was conducted in two phases according to the diameter of the of during the first wave of follicular development at the time of LH challenge (25 mg of pLH). In the first phase, follicles ranging from 5.0 to 6.0 mm (n = 7), 6.1 to 7.0 mm (n = 11), or 7.1 to 8.0 mm (n= 9) were used, and in the second phase, follicles ranging from 7.0 to 8.4 mm (n=10), 8.5 to 10.0 mm (n = 10), or 10.1 to 12.0 mm (n = 9) of diameter were used. After the pLH treatment, the of was monitored by ultrasonography every 12 h for 48 h. No ovulations occurred in heifers in the first phase. However, in the second phase, an effect of follicular diameter was observed on ovulation rate [7.0-8.4 mm (0.0%, 0/10), 8.5-10.0 mm (50.0%, 5/10), and 10.0-12.0 mm (55.6%, 5/9)]. In summary, follicle deviation occurred 2.6d after ovulation in buffalo (B. bubalis) heifers, when the diameters of the DF and SF were 7.2 and 6.4 mm, respectively. No significant changes in plasma concentrations of FSH or LH were detected. Finally, the acquisition of ovulatory capacity occurred when the of reached 8.5 mm in diameter. (C) 2010 Elsevier B.V. All rights reserved. FMVZ USP, Dept Anim Reprod, BR-05508270 São Paulo, Brazil APTA Registro, Registro, SP, Brazil Autonomous Veterinarian, Vicosa, MG, Brazil UFMA, Ctr Ciencias Agr & Ambientais, Chapadinha, MA, Brazil IB UNESP, Dept Bioestatist, Botucatu, SP, Brazil IB UNESP, Dept Farmacol, Botucatu, SP, Brazil Lab Endocrinol UNESP, Dept Apoio & Prod Saúde Anim, Aracatuba, SP, Brazil Univ Saskatchewan, Western Coll Vet Med, Saskatoon, SK S7N 0W0, Canada IB UNESP, Dept Bioestatist, Botucatu, SP, Brazil IB UNESP, Dept Farmacol, Botucatu, SP, Brazil Lab Endocrinol UNESP, Dept Apoio & Prod Saúde Anim, Aracatuba, SP, Brazil FAPESP: 03/10203-4

A total of 177 Nelore heifers were examined by ultrasonography to determine the presence or absence of a corpus luteum (CL) and received a 3 mg norgestomet ear implant plus 2 mg of estradiol benzoate i.m. On Day 8, implants were removed and 150 μg of d -cloprostenol i.m. was administered. At the time of norgestomet implant removal, heifers with or without CL at the time of initiating treatment were assigned equally and by replicate to be treated with 0 IU ( n = 87) or 400 IU ( n = 90) eCG i.m. All heifers received 1 mg of EB i.m. on Day 9 and were submitted to fixed-time artificial insemination (FTAI) 30–34 h later. The addition of eCG increased the diameter of the largest follicle (LF) at FTAI (10.6 ± 0.2 mm vs. 9.5 ± 0.2 mm; P = 0.003; mean ± SEM), the final growth rate of the LF (1.14 ± 0.1 mm/day vs. 0.64 ± 0.1 mm/day; P = 0.0009), ovulation rate [94.4% (85/90) vs. 73.6% (64/87); P = 0.0006], the diameter of the CL at Day 15 (15.5 ± 0.3 mm vs. 13.8 ± 0.3 mm; P = 0.0002), serum concentrations of progesterone 5 days after FTAI (6.6 ± 1.0 ng/ml vs. 3.6 ± 0.7 ng/ml; P = 0.0009), and pregnancy per AI [P/AI; 50.0% (45/90) vs. 36.8% (32/87); P = 0.04]. The absence of a CL at the beginning of the treatment negatively influenced the P/AI [30.2% (16/53) vs. 49.2% (61/124); P = 0.01]. Therefore, the presence of a CL (and/or onset of puberty) must be considered in setting up FTAI programs in heifers. In addition, eCG may be an important tool for the enhancement of follicular growth, ovulation, size and function of the subsequent CL, and pregnancy rates in progestin-based FTAI protocols in Bos indicus heifers.

The aim of the present study was to compare a synchronization of time of ovulation protocol for fixed-timed embryo transfer (FTET) with the usual administration of a single dose of prostaglandin associated with detection of estrus. Also, the effect of the presence of CL at the beginning of FTET protocol was evaluated. Lactating Holstein cows (n=651) with three previous artificial inseminations were classified according to presence or absence of a corpus luteum (CL). Cows with a CL were randomly assigned to two additional treatments and submitted to embryo transfer after detection of estrus (PGF-Estrus) or FTET (FTET-CL). Cows without CL were allocated to the FTET-NoCL treatment. On a random day of the estrous cycle (Day 0), cows in the PGF-Estrus treatment (n=229) were treated with 150 microg d-cloprostenol (PGF) i.m. followed by detection of estrus from Day 1 through Day 5 after PGF. Embryos were transferred 6-8 days after estrus detection. Cows in the FTET-CL (n=208; presence of CL) and FTET-NoCL (n=214; absence of CL) treatments received a norgestomet ear implant plus 2mg estradiol benzoate (EB) and 50mg progesterone i.m. on Day 0. On Day 8, the implant was removed and 400 IUeCG, 150 microg d-cloprostenol and 1mg estradiol cypionate i.m. were administered. No detection of estrus was performed and Day 10 was arbitrarily considered as the estrus day. Ultrasonographic exams were performed in all recipients and only cows with a single CL> or =15 mm or multiple CL received a fresh or frozen-thawed embryo on Day 17. Pregnancy was diagnosed by ultrasonography at 30 and 60 days of pregnancy. When FTET and PGF-Estrus were compared, the proportion of cows receiving an embryo (recipients transferred-to-treated rate) was greater in the FTET-CL (75.0% (156/208) than in PGF-Estrus (34.5%, 79/229; P

A tiered testing strategy based on a comparative chemical and biological testing program has been developed to evaluate the potential of tobacco processes, ingredients, or other technological developments to change the biological activity that results from burning tobacco. Cast sheet tobacco is a specific type of reconstituted tobacco sheet that can be used in the manufacture of cigarettes. The comparative chemical and biological testing program was used to compare the mainstream smoke and cigarette smoke condensate (CSC) from a Reference cigarette that did not contain cast sheet to that collected from Test cigarettes containing cast sheet at a final blend level of either 10% or 15%. Testing included mainstream cigarette smoke chemistry studies, in vitro studies (Ames assay, sister chromatid exchange assay, and neutral red cytotoxicity assay), and in vivo toxicology studies (13-week rat nose-only inhalation assay and 30-week mouse dermal tumor promotion assay). Certain statistically significant differences were observed in the chemical and biological studies when the Reference cigarette was compared to each of the Test cigarettes. However, when viewed collectively, the chemical and biological studies demonstrated that inclusion of cast sheet up to 15% in the final blend did not increase the inherent biological activity of mainstream cigarette smoke or CSC.

The present study investigated how the timing of the administration of estradiol benzoate (EB) impacted the synchronization of ovulation in fixed-time artificial insemination protocols of cattle. To accomplish this, two experiments were conducted, with EB injection occurring at different times: at withdrawal of the progesterone-releasing (P4) intravaginal device or 24h later. The effectiveness of these times was compared by examining ovarian follicular dynamics (Experiment 1, n=30) and conception rates (Experiment 2, n=504). In Experiment 1, follicular dynamics was performed in 30 Nelore cows (Bos indicus) allocated into two groups. On a random day of the estrous cycle (Day 0), both groups received 2mg of EB i.m. and a P4-releasing intravaginal device, which was removed on Day 8, when 400 IU of eCG and 150 microg of PGF were administered. The control group (G-EB9; n=15) received 1mg of EB on Day 9, while Group EB8 (G-EB8; n=15) received the same dose a day earlier. Ovarian ultrasonographic evaluations were performed every 8h after device removal until ovulation. The timing of EB administration (Day 8 compared with Day 9) did affect the interval between P4 device removal to ovulation (59.4+/-2.0 h compared with 69.3+/-1.7h) and maximum diameter of dominant (1.54+/-0.06 acm compared with 1.71+/-0.05 bcm, P=0.03) and ovulatory (1.46+/-0.05 acm compared with 1.58+/-0.04 bcm, P0.01) follicles. In Experiment 2, 504 suckling cows received the same treatment described in Experiment 1, but insemination was performed as follows: Group EB8-AI48 h (G-EB8-AI48 h; n=119) and Group EB8-AI54 h (G-EB8-AI54 h; n=134) received 1mg of EB on Day 8 and FTAI was performed, respectively, 48 or 54 h after P4 device removal. Group EB9-AI48h (G-EB9-AI48 h; n=126) and Group EB9-AI54 h (G-EB9-AI54 h; n=125) received the same treatments and underwent the same FTAI protocols as G-EB8-AI48 h and G-EB8-AI54 h, respectively; however, EB was administered on Day 9. Conception rates were greater (P0.05) in G-EB9-AI54 h [63.2% (79/125) a], G-EB9-AI48 h [58.7% (74/126) a] and G-EB8-AI48 h [58.8% (70/119) a] than in G-EB8-AI54 h [34.3% (46/134) b]. We concluded that when EB administration occurred at device withdrawal (D8), the interval to ovulation shortened and dominant and ovulatory follicle diameters decreased. Furthermore, when EB treatment was performed 24h after device removal, FTAI conducted at either 48 or 54 h resulted in similar conception rates. However, EB treatment on the same day as device withdrawal resulted in a lesser conception rate when FTAI was conducted 54 h after device removal.

A comprehensive 2-year oral chronic toxicity/carcinogenicity study was conducted with smokeless tobacco using modern toxicological test methods and well-accepted standards. The study included a 1-year interim subgroup to assess toxicity at that intermediate time point. Test groups consisted of a tobacco blend (B) used in snus, and an aqueous tobacco extract of that tobacco blend (E) administered at 0.2, 2, or 5 mg nicotine/kg body weight/day via dosed feed to male and female Wistar Han rats. The dosages were selected to simulate potential exposure in humans ingesting smokeless tobacco or an aqueous extract of smokeless tobacco (the latter intended to simulate a snus extract, to enable bridging these data to snus epidemiology data). The following endpoints were evaluated: clinical observations, body weights, feed consumption (FC), ophthalmic exams, toxicokinetics, clinical pathology, gross pathology, and histopathology. During the 2-year study, clear treatment-related, dose-responsive effects included: (1) increases in plasma nicotine and cotinine (indicating that animals were appropriately exposed to levels relevant to human exposure) and (2) decreases in body weights with some alterations in FC. At the 2-year time point, two tumor types (in the highest B doses) displayed statistically significantly increased incidence trends vs. controls: (1) uterine carcinoma in females and (2) epididymal mesothelioma in males. Three tumor types displayed statistically significantly decreased incidence trends: (1) mammary gland adenomas in females, (2) skin basal cell carcinomas in females, and (3) thyroid follicular cell adenomas in males. These increases (and decreases) in tumor trends were interpreted as not being treatment-related because: (1) there were no preneoplastic or related non-neoplastic histopathological findings in the treated rats at the 1-year or 2-year time points to suggest that any of these neoplastic findings were treatment-related and (2) the tumor morphologies and incidences were generally within the expected range of historical controls for Wistar Han rats. Findings from this study indicate that chronic exposure of male and female Wistar Han rats to either a tobacco blend used in snus, or a tobacco extract of that blend does not lead to increased toxicity or carcinogenicity, based on the specified outcomes measured.

A tiered testing strategy has been developed to evaluate the potential for new ingredients, tobacco processes, and technological developments to alter the biological activity that results from burning tobacco. A series of studies was initially conducted with cigarettes containing 3% high fructose corn syrup (HFCS) as an alternate tobacco casing material to corn syrup/invert sugar, including determination of selected mainstream cigarette smoke (MS) constituent yields, Ames assay, sister chromatid exchange (SCE) assay in Chinese hamster ovary (CHO) cells, a 30-week dermal tumor-promotion evaluation of cigarette smoke condensate (CSC) in SENCAR mice, and a 13-week subchronic inhalation study of MS in Sprague-Dawley rats. A second series of studies was conducted with cigarettes containing 3%, 4% and 5% HFCS including MS chemistry, Ames assay, SCE assay in CHO cells, and a neutral red cytotoxicity assays. Collectively, mainstream smoke chemistry, genotoxicity, dermal tumor-promotion, and inhalation toxicity studies demonstrated no differences between cigarettes with 3% HFCS and cigarettes with 3% corn syrup/invert sugar. Also, mainstream smoke chemistry and genotoxicity of cigarettes with 4% and 5% HFCS were not different from cigarettes with 3% HFCS. In conclusion, the addition of up to 5% HFCS to cigarette does not alter the mainstream smoke chemistry or biological activity of mainstream smoke or mainstream smoke condensate as compared to cigarettes with 3% corn syrup/invert sugar with regard to the parameters investigated and presented.

Numerous chemical and toxicological studies indicate that smoke from ECLIPSE, a cigarette that primarily heats rather than burns tobacco, is simplified and reduced in specific chemicals believed to be associated with smoking-related diseases, and demonstrates reduced smoke toxicity and biological activity in vitro when compared to conventional tobacco burning cigarettes. These data led to the hypothesis that cigarette smoke condensate (CSC) from ECLIPSE should have lower tumorigenicity than 1R4F condensate in the SENCAR mouse dermal tumor promotion assay. Female SENCAR mice were initiated with a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) followed by promotion with ECLIPSE or 1R4F CSC. Dermal application of 10, 20, or 40 mg ECLIPSE or 1R4F CSC three times/week for 29 weeks did not alter body weights, survival or other indicators of subchronic toxicity. In DMBA-initiated mice, there were significant increases in both the number of microscopically confirmed tumor-bearing animals and total number of microscopically confirmed dermal tumors at all 1R4F CSC doses and the high-dose ECLIPSE CSC. However, the number of ECLIPSE tumor-bearing animals were reduced 83%, 93% and 67% at the low-, mid- and high-doses, respectively, compared to the 1R4F. Similarly, the total number of dermal tumors was reduced 91%, 94% and 87% at the low-, mid- and high-dose, respectively, compared to the 1R4F CSC. ECLIPSE CSC demonstrated dramatic reductions in dermal tumor promotion potential compared to 1R4F CSC.

A subchronic, nose-only inhalation study compared the effects of mainstream smoke from a 1R4F research cigarette to that of a 2R4F research cigarette. Male and female rats were exposed for 1 h/day, 5 days/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, pulmonary plethysmography, gross pathology, and histopathology were determined. When histological changes resulting from exposure to smoke from the two types of cigarettes were compared, no biologically significant differences were observed. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the 1R4F and the 2R4F cigarettes. The complete toxicological assessment in this comparative inhalation study of 1R4F and 2R4F cigarettes suggests no overall biologically significant differences between the rats exposed to the two cigarettes.

A subchronic, nose-only inhalation study compared the effects of mainstream smoke from a cigarette containing 100% flue-cured tobacco cured by a direct-fired process to that of a cigarette containing 100% flue-cured tobacco cured by a heat exchanger process. The tobaccos and mainstream smoke from tobaccos cured by the heat exchanger process have been shown to have significantly lower levels of tobacco-specific nitrosamines than tobaccos cured by a direct-fired process. Male and female rats were exposed for 1 h/day, 5 days/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. When histologic changes resulting from exposure to smoke from the two types of cigarettes were compared, the only significant difference was increased epithelial hyperplasia of the anterior nasal cavity in males in the high-exposure group for the heat-exchanger cigarette. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the heat-exchanger-cured tobacco cigarette when compared to the direct-fired cured tobacco cigarette. The complete toxicological assessment in this study of heat exchanger and direct-fired tobaccos suggests no overall biologically significant differences between the two cigarettes.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice following a single intraperitoneal (i.p.) injection. However, inhalation of mainstream cigarette smoke does not induce or promote NNK-induced lung tumors in this mouse strain purported to be sensitive to chemically-induced lung tumorigenesis. The critical events for NNK-induced lung tumorigenesis in A/J mice is thought to involve O(6)-methylguanine (O(6)MeG) adduct formation, GC--AT transitional mispairing, and activation of the K-ras proto-oncogene. The objective of this study was to test the hypothesis that a smoke-induced shift in NNK metabolism led to the observed decrease in O(6)MeG adducts in the lung and liver of A/J mice co-administered NNK with a concomitant 2-h exposure to cigarette smoke as observed in previous studies. Following 2 h nose-only exposure to mainstream cigarette smoke (600 mg total suspended particulates/m(3) of air), mice (n=12) were administered 7.5 micromol NNK (10 microCi [5-3H]NNK) by i.p. injection. A control group of 12 mice was sham-exposed to HEPA-filtered air for 2 h prior to i.p. administration of 7.5 micromol NNK (10 microCi [5-3H]NNK). Exposure to mainstream cigarette smoke had no effect on total excretion of NNK metabolites in 24 h urine; however, the metabolite pattern was significantly changed. Mice exposed to mainstream cigarette smoke excreted 25% more 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) than control mice, a statistically significant increase (P0.0001). Cigarette smoke exposure significantly reduced alpha-hydroxylation of NNK to potential methylating species; this is based on the 15% reduction in excretion of the 4-(3-pyridyl)-4-hydroxybutanoic acid and 42% reduction in excretion of 4-(3-pyridyl)-4-oxobutanoic acid versus control. Detoxication of NNK and NNAL by pyridine-N-oxidation, and glucuronidation of NNAL were not significantly different in the two groups of mice. The observed reduction in alpha-hydroxylation of NNK to potential methylating species in mainstream cigarette smoke-exposed A/J mice provides further mechanistic support for earlier studies demonstrating that concurrent inhalation of mainstream cigarette smoke results in a significant reduction of NNK-induced O(6)MeG adduct formation in lung and liver of A/J mice compared to mice treated only with NNK.

The efficiency of different doses of rBST in a MOET protocol was verified in buffaloes. The animals received an intravaginal progesterone device (DIB) plus 2.0 mg of estradiol benzoate (Ric-BE, i.m.) and 0 mg, 250 mg or 500 mg of rBST. Ovarian follicular growth was stimulated by treatment with 200 mg of FSH in decreasing amount (12/12hs). Buffaloes received injections of PGF2α on D6 and on D7. The DIB was removed on D7. On D8, 24 hours after DIB removal, ovulation was induced with GnRH. The follicular emergence and the response to FSH treatment were evaluated by ultrasound on D4 and D8. The artificial insemination (AI) was performed 12 and 24 hours after the GnRH administration. The embryonic structures were collected 5.5 days after the first AI. Data were analyzed by ANOVA. No statistical differences were found between groups. The results indicated that rBST, associated to a MOET protocol at the different dosages used, does not to improve the efficiency of the technique in buffalo.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice, following a single intraperitoneal (ip) injection. However, inhalation of tobacco smoke has not induced or promoted tumors in these mice. NNK-induced lung tumorigenesis is thought to involve O 6 -methylguanine (O 6 MeG) formation, leading to GC→AT transitional mispairing and an activation of the K-ras proto-oncogene in the A/J mouse. NNK can be metabolized by several different cytochromes P 450 , resulting in a number of metabolites. Formation of the promutagenic DNA adduct O 6 MeG is believed to require metabolic activation of NNK by cytochrome P 450 -mediated α-hydroxylation of the methylene group adjacent to the N-nitroso nitrogen to yield the unstable intermediate, methanediazohydroxide. Nicotine, cotinine (the major metabolite of nicotine), and aqueous cigarette tar extract (ACTE) have all been shown to effectively inhibit metabolic activation of NNK to its mutagenic form, most likely due to competitive inhibition of the cytochrome P 450 enzymes involved in α-hydroxylation of NNK. The objective of the current study was to monitor the effects of cotinine and cigarette smoke (CS) on the formation of O 6 MeG in target tissues of mice during the acute phase of NNK treatment. To test the effect of cotinine, mature female A/J mice received a single intraperitoneal injection of NNK (0, 2.5, 5, 7.5, or 10 μmole/mouse) with cotinine administered at a total dose of 50 μmole/mouse in 3 separate ip injections, administered 30 min before, immediately after, and 30 min after NNK treatment. To test the effect of whole smoke exposure on NNK-related O 6 MeG formation, mice were exposed to smoke generated from Kentucky 1R4F reference cigarettes at 0, 0.4, 0.6, or 0.8 mg wet total particulate matter/liter (WTPM/L) for 2 h, with a single ip injection of NNK (0, 3.75, or 7.5 μmole/mouse) midway through the exposure. Cigarette smoke alone failed to yield detectable levels of O 6 MeG. The number of O 6 MeG adducts following ip injection of NNK was significantly (p < 0.05) reduced in both lung and liver by cotinine and by cigarette smoke exposure. Our results demonstrate that NNK-induced O 6 MeG DNA adducts in A/J mice are significantly reduced when NNK is administered together with either cotinine, the major metabolite of nicotine, or the parental complex mixture, cigarette smoke.

Evaluation of the Genotoxic and Cytotoxic Potential of Mainstream Whole Smoke and Smoke Condensate from a Cigarette Containing a Novel Carbon Filter. Bombick, D. W., Bombick, B. Reed, Ayres, P. H., Putnam, K., Avalos, J., Borgerding, M. F., and Doolittle, D. J. (1997). Fundam. AppL Toxicol. 39, 11-17. A novel carbon filter has been developed which primarily reduces the amount of certain vapor phase constituents of tobacco smoke with greater efficiency than the charcoal filters of cigarettes currently in the market In vitro indicators of genotoxic and cytotoxic potential were used to compare the cigarette smoke condensate (particulate phase) or whole cigarette smoke (vapor phase and particulate phase) from cigarettes containing the novel carbon filter with smoke condensate or whole smoke from commercial or prototype cigarettes not containing the novel carbon filter. Ames bacterial mutagenicity, sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells, and neutral red cytotoxicity assays in CHO cells were utilized to assess the genotoxic and cytotoxic potential of the cigarette smoke condensates. SCE and neutral red cytotoxicity assays were utilized to assess the genotoxic and cytotoxic potential of the whole smoke. As expected, the novel carbon filter did not significantly affect the genotoxic or cytotoxic activity of the smoke condensate, although we did observe that the use of low-nitrogen tobacco reduced the mutagenicity of the condensate in Salmonella typhimurium strain TA98. However, the whole smoke from cigarettes containing the novel carbon filter demonstrated significant reductions in genotoxic and cytotoxic potential compared to cigarettes without the novel carbon filter. The toxicity of the smoke was correlated (r = 0.7662 for cytotoxicity and r — 0.7562 for SCE induction) to the aggregate mass of several vapor phase components (acetone, acetaldehyde, acrolein, acrylonitrile, 1,3-butadiene, ammonia, NO,, HCN, benzene, isoprene, and formaldehyde) in the smoke of the cigarettes utilized in this study. In conclusion, this novel carbon filter, which significantly reduced the amount of carbonyls and other volatiles in mainstream cigarette smoke, resulted in significant reductions in the genotoxic and cytotoxic activity of the smoke as measured by

Assessment of the cytotoxicity of mainstream and sidestream cigarette sm oke has traditionally involved exposure of cell cultures to the particulate m atter of smoke. For a m ore com plete assessment of cigarette sm oke cytotoxicity, a technique (cellular smoke exposure technique or CSET) was developed to directly expose mammalian cell cultures to either whole mainstream or sidestream cigarette sm oke. The objective of this study was to com pare the cytotoxicity of whole smoke or vapor phase from mainstream or sidestream sm oke of three Kentucky reference cigarettes. The cigarettes com pared were a high ''tar''cigarette (2R1), a low ''tar'' cigarette (1R4F), and an ultra low ''tar'' cigarette (1R5F). Cytotoxicity was assessed in two cell types (WB rat liver cells and CHO cells) using the neutral red cytotoxicity assay. The order of cytotoxicity of m ainstream smoke from the three cigarettes expressed on a per cigarette basis was 2R1 > 1R4F > 1R5F. Sidestream sm oke from all three reference cigarettes was ...

The genotoxic potential of mainstream smoke from a test cigarette (TOB-HT) that primarily heats tobacco and a representative tobacco-burning cigarette (Kentucky reference 1R4F) was compared in male B6C3/F1 mice after nose-only inhalation exposure. Mice were exposed 1 hr per day, 5 days/ week for a 4 week period to mainstream smoke at concentrations of 0, 0.16, 0.32, and 0.64 mg total particulate matter/liter of air. Micronuclei formation in bone marrow and peripheral blood polychromatic erythrocytes (PCE) of animals exposed to either the TOB-HT or 1R4F cigarette was not significantly different compared with control animals exposed nose-only to filtered and humidified air (sham controls). DNA adduct measurement by the 32 P-post-labeling method indicated an exposure-dependent increase in lung adducts of animals exposed to 1R4F cigarette smoke at all three concentrations with the mid and high exposure groups exhibiting statistically significant increases (P < 0.05) in adduct formation compared to sham-exposed animals. The concentration of DNA adducts in the lungs of animals exposed to the TOB-HT cigarette was not significantly increased (P < 0.05) at any concentration compared to sham-exposed controls. A statistically significant (P < 0.05) concentration-dependent formation of DNA adducts was also observed in the heart tissues of animals exposed to smoke from the 1 R4F cigarette at all three concentrations, but no significant increase in adduct formation was observed in heart DNA of the animals exposed to the TOB-HT cigarette (P < 0.05). Under the conditions of this experiment, the mainstream smoke from the TOB-HT cigarette was demonstrated to be less genotoxic in mice than mainstream smoke from the 1 R4F cigarette, which is representative of cigarettes in the current U.S. market.

An inhalation system was designed to expose experimental animals to aged and diluted sidestream smoke (ADSS), used as a surrogate for environmental tobacco smoke (ETS). The construction of the smoke generator and of the smoke dilution systems is described. Target ADSS concentrations in a 90-day inhalation study were 0.1, 1, and 10 mg/m3 of respirable suspended particulates (RSP). Data is presented on the physical and chemical composition of the smoke presented to animals at or near these target RSP concentrations. The design of the inhalation laboratory was shown to result in highly reproducible respirable aerosols that were effective surrogates of ETS.

Several approaches are available for estimating the caloric availability of fats and fat substitutes. The method described here is a simple bioassay based on the growth of rats as a linear function of caloric intake from either a control fat such as corn oil or a fat of unknown caloric content. Excellent linear responses were found in the 14-day assay, and tests with cocoa butter, lard, and tallow showed agreement with previously reported values. The method is recommended as a simple method for estimation of the caloric value of fats

This study was performed to evaluate the use of an alternate process (using hydrochlorofluorocarbon-123, HCFC-123) for expanding tobacco instead of the standard expansion process (using chlorofluorocarbon-11, CFC-11). Rats were exposed to smoke 1 hr/day, 5 days/week, for 13 weeks. The effects of smoke exposure, primarily on respiratory physiology and on histopathology of the respiratory tract, were as previously reported in the literature. No novel changes were noted. An independent review was made of the above conclusions, formed by the study pathologist. The conclusions made by the reviewing pathologist agreed with those made by the study pathologist. The data obtained in this inhalation study show that there are no differences between the responses noted in animals exposed to smoke from cigarettes containing tobacco expanded with HCFC-123 or with CFC-11.

This manuscript presents data from 90-day toxicology studies designed to characterize the subchronic effects of a smokeless tobacco blend and an aqueous extract of that blend when administered to rodents in NTP-2000 feed. Positive control (nicotine tartrate) and treatment groups were matched for a range of nicotine levels. The doses evaluated were 0.3, 3, and 6 mg nicotine/kg body weight/day in Wistar Hannover rats and 6, 60, and 120 mg nicotine/kg/day in CD-1 mice. Variables evaluated included plasma nicotine and cotinine, body weights, feed consumption, clinical observations, clinical and anatomic pathology (including organ weights), and histopathology. Plasma nicotine and cotinine levels were dose-responsive. Key effects such as body weight reductions and organ weight changes occurred in rats and mice predominantly at the highest doses of test articles and positive control in the absence of treatment-related gross or histopathological changes. Organ weight changes were attributed mainly to the lower body weights of treated vs. control groups. The blend- and extract-induced effects generally paralleled each other and the nicotine-induced effects. Based on these studies, the doses evaluated spanned the no observable adverse effect level, the lowest observable adverse effect level and the maximum tolerated dose.

Sprague-Dawley rats were exposed 6 hr per day for 14 consecutive days to aged and diluted sidestream smoke (ADSS), used as a surrogate for Environmental Tobacco Smoke (ETS), at concentrations of 0.1 (typical), 1 (extreme), or 10 (exaggerated) mg of particulates per cubic meter. Animals were exposed nose-only, inside whole-body chambers, to ADSS from the 1R4F reference cigarette. End-points included histopathology, CO-oximetry, plasma nicotine and cotinine, clinical pathology, and organ and body weights. The only pathological response observed was slight to mild epithelial hyperplasia and inflammation in the most rostral part of the nasal cavity, in the high-exposure group only. No effects were noted at medium or low exposures. The minimal changes noted were reversible, using a subgroup of animals kept without further treatment for an additional 14 days. Overall, the end-points used in the study demonstrated that there was no detectable biological activity of ADSS at typical or even 10-fold ETS concentrations and that the activity was only minimal at very exaggerated concentrations (particle concentrations 100 times higher than typical real-world concentrations).

Relative sensory irritation responses for Swiss-Webster mice exposed nose-only to mainstream tobacco smoke were evaluated for several cigarette types using a smoking regimen consisting of a 35-ml puff, 2 s in duration, taken once per minute. The degree of sensory irritation for each cigarette type was evaluated as the smoke concentration inducing a 50% reduction in breathing frequency. The smoke concentration inducing 50% respiratory depression is called the RD(50) value. Study findings suggest that mainstream tobacco smoke from the Eclipse cigarette, which primarily heats rather than burns tobacco, yielded an RD(50) that was significantly higher (approximately twofold) than a tobacco-burning leading ultralight or the 2R4F or 1R5F reference cigarettes. This is indicative of reduced upper airways irritation by Eclipse that may be due to its distinct design. Study findings suggest that the irritating nature of mainstream tobacco smoke from different cigarette types can be evaluated effectively in terms of smoke concentration using the relative sensory irritation assessment. These findings constitute the first report about use of the RD(50) sensory irritation response during comparative evaluations of mainstream tobacco smoke.

Summary: Nicotine in smoke presented to animals in inhalation studies is currently determined by placing glass-fiber pads (“Cambridge“ pads) in unoccupied animal ports in the smoking machine and pulling a fixed volume of smoke through the pad. The nicotine is extracted from the pad and quantified using gas chromatography (GC). A near-infrared (NIR) instrument was used to develop an alternative technique, which provides estimates of nicotine concentrations on Cambridge pads in only 1–2 min. For given cigarette types, the NIR data are highly correlated with the GC data. This new method results in considerable time-saving during inhalation studies with cigarette smoke.

The induction of sustained, chemically-induced regenerative hyperplasia during rodent bioassays may result in carcinogenesis. Hence, accurate measurement of replicative DNA synthesis and cell proliferation in putative target organs during acute and subchronic assays will aid in dose selection for chronic studies; and assessment of cell proliferation rates during chronic bioassays will aid in the interpretation of results and in risk assessment. Histopathology is the traditional method for assessing hyperplasia and will continue to be of paramount importance. However, quantitative molecular approaches for assessing rates of DNA synthesis and mitogenesis in many cases extend and complement histopathological examinations. Three of these methodologies, incorporation of radiolabeled thymidine into DNA, incorporation of bromodeoxyuridine into DNA, and measurement of proliferating cell nuclear antigen, are discussed in this review. Also, a relatively new technology, the use of flow cytometry to measure the incor...

The genotoxic effects of 90-day nose-only exposures to smoke from new cigarettes, which heat but do not burn tobacco (New), or from reference cigarettes, which burn tobacco, were evaluated in Sprague-Dawley rats by examining the cytogenetic endpoints of sister-chromatid exchanges (SCE), chromosome aberrations, and micronuclei in bone-marrow cells. The concentrations of wet total particulate matter (WTPM) and carbon monoxide in the smoke from both cigarette types were similar. The mainstream smoke from both New and reference cigarettes was adjusted to WTPM concentrations of approx. 200 and 400 μg/1 for low and high smoke exposure. Rats were exposed to smoke 1 h per day, 5 days per week for 13 consecutive weeks. Inhalation of smoke by the exposed animals was confirmed by analysis of blood carboxyhemoglobin and plasma nicotine. Examination of bone-marrow cells following the final day of exposure showed that smoke from neither the New nor reference cigarette induced a positive response in the SCE, chromosome aberration, or micronucleus assays in rats.

Male and female B6C3F1 mice were exposed nose-only to smoke from a test cigarette that heats tobacco, or from a reference cigarette that burns tobacco. Cigarette smoke was generated by a smoking machine, and the concentrations of wet total particulate matter (WTPM) were adjusted to 0, 0.16, 0.32, or 0.64 mg/l. Exposures were performed 1 h/day for 14 consecutive days. Urine mutagenicity was assessed by a modified Ames bacterial assay Clastogenesis (sister-chromatid exchanges, chromosome aberrations, and micronuclei) was evaluated in bone marrow cells. Respiratory rate was depressed significantly by exposure to smoke from the reference cigarette, but not the test. Blood carboxyhemoglobin, plasma nicotine, and plasma cotinine showed exposure-dependent increases in the smoke-exposed animals. Histopathological changes similar to those noted previously in smoke-exposed rats were noted, with fewer and less pronounced changes in the animals exposed to smoke from the test cigarette when compared with the r...

A tiered testing strategy has been employed to evaluate the potential for new ingredients, tobacco processes, and technological developments to alter the mainstream smoke or biological activity that results from burning cigarette tobacco. The foundation of this evaluation strategy is comparative testing, typically including chemical and biological assessments. In the manufacture of cigarettes, diammonium phosphate (DAP) and urea have been historically used as ingredients added to tobacco, to reconstituted tobacco sheet, and to other processed tobaccos. As part of ongoing stewardship efforts, a toxicological assessment of cigarettes with and without DAP and urea was conducted. Chemical and biological analyses were conducted for test cigarettes added 0.5% DAP and 0.2% urea in the final blend and also for those added 1.0% DAP and 0.41% urea in the final blend compared to reference cigarettes without added DAP or urea. Principal components of this evaluation included a determination of selected mainstream smoke constituent yields, an Ames assay in Salmonella typhimurium strains TA98 and TA100, a sister chromatid exchange assay in Chinese hamster ovary cells, a 13-week inhalation study of mainstream cigarette smoke in Sprague-Dawley rats, and a 30-week dermal tumor-promotion evaluation of mainstream cigarette smoke condensate in SENCAR mice. Comparative evaluations demonstrated that the addition of DAP and urea to cigarettes at up to 1% and 0.41%, respectively, does not alter the biological activity compared to reference cigarettes without DAP or urea.

A subchronic, nose-only inhalation study was conducted to compare the effects of mainstream smoke from a reference cigarette containing conventional reconstituted tobacco sheet at 30% of the finished blend to mainstream smoke from cigarettes containing 10% or 15% cast sheet (a specific type of reconstituted tobacco sheet) substituted for part of the conventional reconstituted tobacco. Male and female Sprague-Dawley rats were exposed for 1 h/day, 5 d/wk, for 13 wk to mainstream smoke at 0, 0.06, 0.20, or 0.80 mg wet total particulate matter per liter of air. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin (COHb), serum nicotine, plethysmography, gross pathology, and histopathology were determined. Exposure to cigarette smoke induced a number of changes in respiratory physiology, histopathology, and serum nicotine and COHb levels when compared to sham animals. When corresponding dose groups of reference and cast sheet mainstream smokes were compared, no biological differences were noted. At the end of the exposure period, subsets of rats from each group were maintained without smoke exposures for an additional 13 wk (recovery period). At the end of the recovery period, there were no statistically significant differences in histopathological findings observed between the reference and either cast sheet cigarette. Substitution of 10% or 15% cast sheet tobacco for conventional reconstituted tobacco sheet does not alter the inhalation toxicology of the mainstream smoke when compared to mainstream smoke from a reference cigarette containing conventional reconstituted tobacco sheet.

This study compared the toxicological responses of Sprague-Dawley rats exposed nose-only to mainstream smoke (MS) from Test cigarettes (1, 2, and 3) to those of Control cigarettes without banded cigarette paper technologies (BCPT). Test cigarettes 1 and 2 had bands based on one technology (different band weight application) while Test cigarette 3 had bands based on another technology. The banded papers are representative of current marketed technologies. Rats were exposed to humidified HEPA filtered air (Sham) or to MS at concentrations of 0.06, 0.20, or 0.80 mg wet total particulate matter per liter air. Each exposure group contained 30 animals/sex (sentinel had 20 animals/sex). The study had two phases (13 weeks each): MS exposure (1 h/day, 5 days/week) and recovery without smoke exposure. Endpoints included clinical observations, respiratory physiology, hematology, serum chemistry, blood carboxyhemoglobin (COHb), serum nicotine, body/organ weights, gross pathology, and histopathology. Comparisons conducted were: Sham exposed vs. all cigarettes, Control cigarette vs. all Test cigarettes, and Test 1 vs. Test 2. Control and Test MS had comparable effects on respiratory physiology, COHb, serum nicotine, serum chemistry, and hematology. While some minor differences were observed, Control and Test MS had comparable effects on clinical signs, body/organ weights, and gross pathology/histopathology. Consequently, exposure of rats to equivalent MS concentrations from the four cigarettes induced similar toxicological responses in this study.

A tiered testing strategy has been developed to evaluate the potential of tobacco processes, ingredients, or technological developments to change the biological activity resulting from burning tobacco. The strategy is based on comparative chemical and biological testing. Dry ice expanded tobacco (DIET) is an example of a common tobacco expansion process currently used in the manufacture of cigarettes to increase tobacco filling capacity. As part of the toxicological evaluation of DIET, test cigarettes containing DIET were compared with control cigarettes containing tobacco expanded with a traditional expansion agent (Freon-11, also known as trichlorofluoromethane). Testing included mainstream cigarette smoke chemistry studies, genotoxicity studies (Ames and sister chromatid exchange, SCE), a 13-week inhalation study in Sprague-Dawley rats, and a 30-week dermal tumor promotion study in SENCAR mice. Cigarettes containing DIET or Freon-11 expanded tobacco were similar in biological activity.

A subchronic, nose-only inhalation study comparing the potential biological activity of mainstream smoke from a cigarette that primarily heats tobacco (Eclipse) to mainstream smoke from a 1R4F reference cigarette was conducted using Sprague-Dawley rats of each gender. Smoke exposures were for 1 h/day, 5 days/wk for 13 wk, at concentrations of 0, 0.16, 0.32, or 0.64 mg wet total particulate matter (WTPM)/L air. Smoke was generated at the Federal Trade Commission standard of a 2-s puff of 35 ml, taken once per minute. Clinical signs, body and organ weights, clinical chemistry, hematology, carboxyhemoglobin, serum nicotine, plethysmography, gross pathology, and histopathology were determined. Plethysmography indicated that respiratory rate was decreased at all concentrations of 1R4F smoke, but only at the high concentration of Eclipse smoke. Tidal volume was depressed and minute volume was lower for all smoke-exposed rats. Rats exposed to Eclipse smoke inhaled more smoke at the low and mid-concentration exposures than rats exposed to equivalent concentrations 1R4F smoke. Carboxyhemoglobin and serum nicotine were directly related to the exposure concentrations of carbon monoxide (CO) and nicotine in an exposure-dependent manner. Body weights were slightly lower in smoke-exposed rats, while no treatment-related effects were seen in clinical signs, clinical chemistry, hematology, or gross changes at necropsy. The only treatment-related effect seen in organ weights was an increase in heart weight in females in the Eclipse high-concentration exposure group, attributed to higher CO in the Eclipse exposure atmosphere. Higher CO resulted from the lower dilution of Eclipse smoke required to maintain WTPM concentrations equal to those of the 1R4F smoke, and not from a higher CO yield from Eclipse cigarettes. Nasal epithelial hyperplasia and ventral laryngeal squamous metaplasia were noted after exposure to either the 1R4F or Eclipse smoke. The degree of change was less in Eclipse smoke-exposed rats. Lung macrophages were increased to a similar extent in the Eclipse and 1R4F smoke-exposed groups. Brown/gold pigmented macrophages were detected in the lungs of rats exposed to 1R4F smoke, but not those exposed to Eclipse smoke. Subsets of rats from each group were maintained for an additional 13 wk without smoke exposures. Most of the changes noted at the end of the smoke exposures had disappeared, while those that remained were regressing toward normal. Evaluation of these findings indicated the overall biological activity of Eclipse smoke was less than 1R4F smoke at comparable exposure concentrations.

This study was designed to determine if a prototype of the Eclipse cigarettes, which uses a special form of continuous glass filament (CGF) as an insulator around the carbon heat source, yielded CGFs via mainstream smoke. A method was developed that used electrostatic precipitation with a greater than 99% collection efficiency of mass to capture CGFs transferred to mainstream smoke. The cigarettes were smoked using an exaggerated puffing condition that was more than twice the Federal Trade Commission (FTC) standard. The cigarettes were subjected to handling procedures that simulated commercial shipping conditions prior to smoking. CGFs were intentionally added to a series of smoke condensate samples to determine CGF recovery efficiency. The recovery efficiency was determined for a series of four internal standards added to smoke condensate. The recovery efficiency was 86% for the Eclipse 5-014 prototype. The number of CGFs in smoke condensate collected from the Eclipse 5-014 prototype was approximately 0.06 +/- 0.02 CGFs per cigarette (+/- standard deviation), including the background counts of CGFs and 0.03 CGFs per cigarette, when corrected for background contributions. The number of CGFs found in smoke condensates for this prototype was not statistically distinguishable from zero or background in these experiments, which were capable of detecting transfer rates of greater than 0.2 CGFs per cigarette.

Mainstream smoke from Kentucky reference low “tar” (1R4F) and ultra-low “tar” (1R5F) cigarettes and a test cigarette (TOB-HT), that primarily heats tobacco, was compared for cytotoxic and genotoxic potential using cellular smoke exposure technology (CSET). CSET includes a computer controlled 30-port AMESA/Battelle-Geneva smoke generator which exposes cultured mammalian Chinese hamster ovary cells (CHO) to whole smoke. Cytotoxicity was assessed using the neutral red assay and genotoxicity was assessed using the sister chromatid exchange (SCE) assay. Compared on a per cigarette basis, mainstream smoke from 1R5F and the TOB-HT cigarette was significantly less cytotoxic and genotoxic than the smoke from the 1R4F cigarette. The cytotoxic and genotoxic activity of smoke from the TOB-HT cigarettes was slightly greater than the smoke from the ultra-low “tar” Kentucky 1R5F reference cigarettes. In conclusion, in these assays mainstream whole smoke of the TOB-HT cigarette had slightly greater cytotoxic and genotoxic potential compared with an ultra-low “tar” 1R5F Kentucky reference cigarette and significantly less activity compared with the whole mainstream smoke from a low “tar” 1R4F Kentucky reference cigarette, representative of the US market average cigarette for FTC yields of “tar”, CO and nicotine.

Although histopathology will continue to be essential for assessing the results of rodent inhalation studies, molecular toxicology endpoints are of increasing importance, as these techniques often complement and extend histopathological examinations. One of the primary uses of molecular toxicology is determining the delivered dose of the inhaled material to macromolecules in target tissues. During inhalation studies this is most often done by measuring DNA adducts in the respiratory tract. DNA adducts may be measured specifically (e.g. using monoclonal antibodies or mass spectrometry) or non-specifically (e.g. by using the 32 P-post-labeling assay). Another major use of molecular toxicology techniques is the assessment of cellular and molecular changes in target tissues which may precede or be more sensitive than histopathologic alterations. For example, rates of cellular DNA synthesis occurring in target tissues may be quantified at any time during the study by administering the animals either radiolabelled thymidine or the non-radiolabelled thymidine analog bromodeoxyuridine (BrdU). Pulmonary changes may be assessed in bronchoalveolar lavage fluid using either cellular (e.g. macrophage number, granulocyte number) or biochemical (e.g. alkaline phosphatase, lactate dehydrogenase) techniques. The potential of the inhaled material to produce genetic alterations may be evaluated by examining the chromosomes of pulmonary alveolar macrophages for cytogenetic changes. To illustrate the use of these endpoints, an experiment was conducted to determine the molecular toxicology of aged and diluted sidestream smoke (a surrogate for environmental tobacco smoke) in rodent inhalation studies. The endpoints measured were DNA adducts in target and non-target tissue, chromosome aberrations in pulmonary alveolar macrophages, and DNA synthesis in the epithelial lining of the nasal turbinates. In conclusion, quantitative molecular toxicology techniques are expected to assume an increasingly prominent role in the conduct and interpretation of rodent inhalation studies.

Estradiol given during progestin-treatment promotes emergence of a new follicular wave in cattle. The interval from estradiol treatment to follicular wave emergence is dependent on the estradiol preparation, the dose, and possibly the animal category (cows or heifers). Therefore, this experiment aimed to compare the effects of either estradiol benzoate (EB) or estradiol valerate (EV) on the synchrony of ovarian follicular wave emergence in Bos indicus (Nelore) heifers and cows treated with a Norgestomet implant (Crestar�; Intervet, Boxmeer, The Netherlands; factorial 3 � 2 design). Lactating cows (n = 30; 70-90 days postpartum) and cycling heifers (n = 29) were homogeneously assigned, according to the presence of corpora lutea, to six experimental groups (G2EB, G2.5EV, and G5EV for each animal category). At random stages of the estrous cycle (Day 0), the females received an auricular implant and were assigned to receive 2 mg of EB (Farmavet, S�o Paulo, Brazil; 10 cows and 10 heifers; G2EB), 2.5 mg of EV plus 1.5 mg of Norgestomet (10 cows and 10 heifers; G2.5EV; half of a recommended dose), or 5 mg of EV plus 3 mg of Norgestomet (10 cows and 10 heifers; G5EV, full recommended dose) i.m. Transrectal ultrasonography was performed once daily from Day 0 to confirm the follicular dominance (new growing dominant follicle >8 mm in diameter). The day of wave emergence was retrospectively defined as the day when the dominant follicle was first detected at a diameter of 3 mm. Two-way analysis of variance was used to compare the effects of treatments (G2EB, G2.5EV, and G5EV), category (cows and heifers), and the treatments-by-category interaction on the interval from beginning of the treatment to wave emergence. The LSD was used to locate differences among groups and the Bartlett's test revealed heterogeneity of variance. Statistical analysis was conducted by SAS for Windows (SAS Institute, Inc., Cary, NC, USA). There was a significant effect of treatment and category (P < 0.001), but there were no treatments-by-category interactions (P > 0.05). A new follicular wave was induced in all animals after treatment with EB or EV. The heifers given G2EB (2.50 � 0.19a; 2 to 3x days) had a reduced (P < 0.01) and least variable interval (P < 0.05) than those given G2.5EV (4.20 � 0.27b; 3 to 6xy days) or G5EV (6.10 � 0.57c; 3 to 8y days). The cows given G5EV (4.00 � 0.52b; 2 to 6y days) had the longest (P = 0.05) and more variable interval (P < 0.001) than those given G2EB (2.50 � 0.22a; 2 to 4x days), but were similar to those given G2.5EV (3.10 � 0.41ab; 2 to 6xy days). The effect of EB was similar in heifers and cows (P > 0.05); however, the EV or 2.5EV promoted a longer emergence wave interval in the heifers than in the cows (P < 0.05). Analyzing the main effects, cows and heifers given G2EB (2.50 � 0.15a; 2 to 4x days) had a smaller and less variable interval than those given G2.5EV (3.65 � 0.27b; 2 to 6y) or G5EV (5.05 � 0.44c; 2 to 8z). In conclusion, the EV treatment, in both doses (5 or 2.5 mg), resulted in a delayed and more variable interval to emergence of a new follicular wave than 2 mg of EB in Bos indicus cows and heifers treated with a Norgestomet auricular implant. This work was supported by Intervet, Brazil.

The interaction of formaldehyde (CH/sub 2/O) with reduced glutathione (GSH) was evaluated in aqueous solution and in isolated perfused lungs and livers. Addition of CH/sub 2/O (0-4.9 mM) to a solution of 0.17 mM GSH in 2 mM EDTA, pH 7.4, resulted in a time- and concentration-dependent depletion of GSH. Perfusion of livers with fortified Krebs-Ringer bicarbonate buffer containing 0.3-4.9 mM CH/sub 2/O resulted in a dose-dependent depletion of GSH. Perfusion of isolated ventilated lungs with perfusate containing 4.9 mM CH/sub 2/O resulted in a depletion of GSH to 75% of controls. However, lower concentrations of CH/sub 2/O in the lung perfusate did not result in depletion of GSH. These results demonstrate that exposure ito CH/sub 2/O in aqueous solution or in the perfused lung and liver is capable of depleting endogenous GSH. However, the concentrations of CH/sub 2/O required to yield a significant depletion of endogenous GSH exceed those encountered in vivo. Thus it is unlikely that depletion of GSH by CH/sub 2/O is a causal factor in formaldehyde-induced toxicity.

Ruthenium(III) reacts with 2,2′,2″-terpyridine in aqueous solution at pH 3.0–4.5, when heated at 85 °C for 2 min, giving a green cationic complex with an absorbance maximum at 690 nm. The color is stable for at least 25 h. The system conforms to Beer's law. The optimal range for measurement (1.00-cm optical path) is 2–10 p.p.m. Ru; the molar absorptivity is 8.3 ·103. Ruthenium(II) reacts with terpyridine at pH 5.5 to develop an amber cationic complex (absorption maximum at 475 nm) on heating at 95° C for 45 min. The color is apparently stable indefinitely. The system conforms to Beer's law; the optimal range is 1–5 p.p.m. Ru; the molar absorptivity is 1.45·104 l mol−1 cm−1. Common anions do not interfere; separation as RuO4 is necessary when iron and a few other transition cations are present. The green complex, a strong oxidant, is converted to the ruthenium(II) complex by oxidation of water, slowly at room temperature, or more quickly by longer heating and/or higher temperature, and by increase of pH. The Ru(II) complex can be converted to the Ru(III) complex by strong oxidants such as Ce(IV). In the amber complex, the reaction ratio is 1 Ru: 2 terpyridine, in which the ligand is tridentate, whereas in the green complex the reaction ratio is 1 Ru : 3 terpyridine, the latter acting only as a bidentate ligand. Short gentle warming of a mixture of ruthenium(III) and terpyridine first produces a transient unidentified blue-colored species (absorbance at 790 nm).

Animals were exposed nose-only to 0, 527, 1091, or 1800 parts per million (ppm) carbon monoxide for 1 h/day, for 74 consecutive days. Blood carboxyhemoglobin (COHb) concentrations in the 1800 ppm group approached those concentrations associated with lethality (62+%). Respiratory minute volume was reduced to 73% of preexposure values in animals exposed to 1800 ppm CO, through changes in respiratory rate. Most of the erythrocyte parameters showed increases that were proportional to the CO concentration. Accumulation half-lives for COHb in the low, medium, and high exposure groups were 17.2, 12.6, and 9.0 min, respectively; the elimination half-lives were 42.3, 32.6, and 29.7 min, respectively Heart weights were increased by up to 32% in males and 47% in females as a result of this very high exposure to 1800 ppm CO. Chronic inflammation was observed in the cardiac muscle of animals exposed to 1800 ppm CO. The inflammation consisted of scattered interstitial aggregates of lymphohistiocytic cells, foun...

Azodicarbonamide (ADA) is widely used as a blowing agent in the manufacture of expanded foam plastics, as an aging and bleaching agent in flour, and as a bread dough conditioner. Human exposures have been reported during manufacture as well as during use. Groups of male F344/N rats were administered ADA by gavage, by intratracheal instillation, and by inhalation exposure to determine the disposition and modes of excretion of ADA and its metabolites. At 72 hr following gavage, 30% of the administered ADA was absorbed whereas following intratracheal instillation, absorption was 90%. Comparison between groups of rats exposed by inhalation to ADA to achieve body burdens of 24 or 1230 micrograms showed no significant differences in modes or rates of excretion of (/sup 14/C)ADA equivalents. ADA was readily converted to biurea under physiological conditions and biurea was the only /sup 14/C-labeled compound present in excreta. (/sup 14/C)ADA equivalents were present in all examined tissues immediately after inhalation exposure, and clearance half-times on the order of 1 day were evident for all tissues investigated. Storage depots for (/sup 14/C)ADA equivalents were not observed. The rate of buildup of (/sup 14/C)ADA equivalents in blood was linearly related to the lung content as measuredmore » from rats withdrawn at selected times during a 6-hr inhalation exposure at an aerosol concentration of 25 micrograms ADA/liter. In a study extending 102 days after exposure, retention of (/sup 14/C)ADA equivalents in tissues was described by a two-component negative exponential function. The results from this study indicate that upon inhalation, ADA is rapidly converted to biurea and that biurea is then eliminated rapidly from all tissues with the majority of the elimination via the urine.« less

Dibenzo[c,g]carbazole (DBC) is a nitrogen-containing polycyclic aromatic hydrocarbon that has been detected in tobacco tars, industrial oils, and diesel engine exhaust fumes. DBC is carcinogenic in respiratory tract tissue of hamsters and in lungs, kidneys, and livers of mice. The purpose of this research was to determine the respiratory tract deposition, distribution in tissues, metabolism, and excretion of DBC in rats after inhalation. Rats were exposed nose-only to 1.1 or 13 μg [14C]DBC/liter air for 60 min. Activity median aerodynamic diameters for the two concentrations of DBC ranged from 0.7 to 0.8 μm. Urine, feces, and selected tissues were collected for various times after exposure. The fractional deposition for the 1.1 and 13 μg/liter exposure concentrations was similar, 13 and 16%, respectively. The dominant route of excretion of 14C following exposure to either concentration of DBC was the feces, accounting for approximately 95% of the total 14C eliminated. Half-time for lecal excretion was 20 ± 6 hr ( x ± SE ). Gastrointestinal absorption of [14C]DBC was 43%. Radioactivity was widely distributed to all tissues examined, with the respiratory tract (lung, trachea, larynx, and nasal turbinates), upper gastrointestinal tract (stomach and small intestine), the liver, and the adrenals containing the highest concentrations of [14C]DBC equivalents within 1 hr after exposure. At both concentrations of DBC tested, clearance of 14C from tissues was rapid, with approximately 60 to 98% of the initial tissue burden being cleared with half-times ranging from 1 to 16 hr. The remaining 2 to 40% in the tissues was cleared with half-times that ranged from 1.5 to 14 days. Several metabolites were detected in the urine and feces, none of which appeared to be either glucuronide or sulfate conjugates. Small quantities of [14C]DBC were detected in the urine, although quantities were less than 1% of the initial respiratory tract burden of [14C]DBC. The results from this research indicate that DBC was rapidly absorbed from the lungs and translocated to many tissues. Prior to elimination, primarily in the feces, DBC was extensively metabolized. There appeared to be no effect of exposure concentration on the toxicokinetics of inhaled DBC.

Eight groups of 30 male and 30 female rats were exposed 1 hr per day, 5 days per week for 13 weeks, to smoke from reference (tobacco burned) or test (tobacco only heated) cigarettes, at nicotine concentrations of 5, 15, or 30 micrograms/liter of air. Similar smoke concentrations of wet total particulate matter and carbon monoxide were produced in each of the test/reference comparisons. There was a pronounced depression of minute ventilation of animals in the reference groups, but not in the test animals. Blood carboxyhemoglobin concentrations were similar in animals exposed to smoke from test and reference cigarettes. Plasma concentrations of nicotine and cotinine in the test groups were higher than in the reference groups. There were no differences between the smoke-exposed groups in terms of body weight or feed consumption. At necropsy, an increase in heart weight was noted in both high exposure groups. There were notable differences in histopathology, with fewer and less-pronounced changes in the test groups than in the reference groups. Many of the histopathological responses induced in the reference groups were absent in the test groups. Overall, the study demonstrated a substantial reduction in the biological activity of smoke from the test cigarette when compared with the reference.

Ruthenium(II) reacts with 3-(2-pyridyl)-5.6-diphenyl-1,2.4-triazine (PDT) in water-alcohol solution at pH 5 to give a magenta-colored cationic complex having an absorption peak at 485 nm. The color is fully developed by heating at 85° C for 30 min. The system conforms to Beer's law; the optimal concentration range for measurement at 1.00-cm optical path is about 0.5–3.4 p.p.m. of ruthenium. The molar absorptivity is 2.1 · 10 4 1 mole -1 cm -1 . The effects of reagent concentration, heating temperature and time, pH, and alcohol concentration have been studied. Common anions do not interfere. Separation from interfering cations is effected by distillation and recovery of ruthenium tetroxide. Spectrophotometric methods of continuous variations and of mole ratios, and elemental analysis of solid salts isolated from solution, indicate a ruthenium-to-PDT stoichiometry of 1:2, the PDT acting as a tridentate ligand.

Reaction velocities at various temperatures have become of much interest to biologists, on account of the possibility they present for the identification of certain physiological processes as chemical or physical, since it has been found that certain common physical and chemical reactions have rather characteristic temperature coefficients. Thus, if we suspect that a certain reaction is due to a chemical process, we can determine whether or not the velocity of the reaction at various temperatures is of the magnitude demanded by a chemical reaction. Since the temperature coefficients for all chemical reactions do not always have the same order of magnitude as demanded by the van't Hoff rule on the dependence of the rate of reaction of chemical processes upon temperature (9), the test is not wholly conclusive, but indicates rather a probable explanation of the phenomenon in question. Animal physiologists in recent years have given much attention to temperature coefficients, but very little work has been done upon this subject by plant physiologists. CLAUSEN (2) was the first to show that the rate of evolution of carbon dioxide from seedlings and buds was about doubled with an increase of io? C. M\IiSS MATTHAEI (5) found that van't Hoff's rule applied to the fixation of carbon dioxide by leaves in sunlight and to the evolution of this gas by leaves in the dark. PRICE (7) determined the temperature coefficient for the opening of flower buds, and found it to have the magnitude demanded for a chemical reaction. It will be seen that these experiments deal with growth phenomena. LOEB (4) was the first to call attention to the fact that the temperature coefficient of the duration of life of sea urchin eggs differs widely from that of their development, the temperature coefficient of development being 2.86 for a rise in temperature of Io' C., while that of duration of life is nearly 2 for a rise in temperature of i? C. MOORE (6) found that the temperature 65] [Botanical Gazette, vol. 62

A sensitive spectrophotometric determination of osmium is based on the blue color (absorption maximum at 615 mμ formed by reaction of osmium with p-(morpholino)-N-(4'-hydroxy-3'-methoxy)benzylidineaniline (“anil”) in acetate-buffered solution containing ethanol to prevent formation of a precipitate, Full color development is attained in I h at room temperature, and the color is stable for several hours. The absorbance is reproducible. The optimum concentration range for I-cm optical path is about I to 4 p.p.m. of osmium. Several transition elements interfere ; osmium can be separated as its tetroxide by the usual distillation method. The blue product is a cationic complex formed by reaction of anil with osmium in a 2 : I mole ratio. When osmium is in excess a red cationic complex (absorption maximum at 466 mμ) is formed by a I : I reaction between osmium and the reagent. The I : I complex is slowly converted to the 2 : I complex by excess reagent.

A spectrophotometric determination of osmium has been developed, based on the purple color (absorption maximum at 515 mμ) formed by reaction of osmium with quinisatin oxime in buffered solution of dimethyl formamide and methanol. The absorbances are reproducible, and the system conforms to Beer's law. The method compares favorably in sensitivity with existing methods for osmium. The optimum concentration range (for 1 cm optical path) is about 2 to 10 p.p.m. of osmium. Although the maximum color develops slowly, it is stable for 7 days or longer. Several elements, notably iron, cobalt, and ruthenium, interfere, so that separation, is necessary. A reaction ratio of 1:2 for osmium and quinisatin oxime was clearly indicated; some evidence was also obtained for the presence of higher complexes.

A rapid, reliable method for the analysis of mercury(I) salts is based on direct weighing of mercury formed by complete disproportionation of Hg22+, accomplished by complexing the mercury(II) with bromide or iodide. The free mercury is obtained in the form of a bright metallic globule, which is easily washed and dried for weighing. The method has been applied to the analysis of mercury(I) chloride and mercury(I) nitrate, and has been compared with titrimetric methods involving oxidation of mercury(I) with iodate and with iron(III).

Platinum(IV or II) reacts with o-phenylenediamine, at pH 6.5, to form light blue solutions having maximum absorption at 703 nm. The reaction, slow at room temperature, is complete in 3-4 min at temperatures near 100 degrees . Use of dimethylformamide in the solution prevents precipitation of the reaction product. The colour is stable for at least 24 hr. Effects of heating temperature and time, pH, reagent concentration, and other variables have been studied. The system conforms to Beer's law over the concentration range investigated. Optimum concentration range for measurement in 10-mm cells is 0.4-1.4 ppm of platinum; the molar absorptivity is 9.83 x 10(3) l. mole(-1). mm(-1). Interference from foreign ions has been evaluated, and methods for removal or masking of interferences have been tested. In the colour-forming reaction, platinum(IV) is reduced by the organic reagent to platinum(II), which is then complexed with the reagent. The metal : ligand ratio of 1 : 2 was indicated by solution spectrophotometric measurements, and was confirmed on the solid reaction product by elemental analysis and by mass spectrometric examination.