The progression of hepatocellular adenomas to carcinomas has been less well documented in mice th... more The progression of hepatocellular adenomas to carcinomas has been less well documented in mice than in rats. We studied progression of spontaneous and chemically induced hepatocellular adenomas in male C3H/HeNCr mice by image analysis. Spontaneous lesions in 15, 18 and 21 month old untreated male C3H/HeNCr mice and experimentally induced lesions were examined. Experimental group 1 received a single i.p. injection of N-nitrosodiethylamine (DEN) (5 mg/kg body wt) at 15 days of age. Groups 2 and 3 were injected a second time with DEN at 15 or 20 weeks of age (75 mg/kg body wt), with interim sacrifices at 11, 16 and 34 weeks after the second DEN injection. Atypia in adenomas were classified into four grades according to cell size, tinctorial changes, cellular pleomorphism and trabecular pattern. At earlier stages of the neoplastic process (11 or 16 weeks after the second DEN dose), most adenomas were well-differentiated lesions with no atypia or focal grade 1 or 2 atypia. At later stages (34 weeks after the second DEN dose), a large proportion of hepatocellular tumors were classified as adenoma with grade 3 atypia or carcinoma. The proportion of carcinomas in mice treated with a second dose of DEN at 20 weeks of age was significantly higher than in mice treated with a single dose of DEN or in mice given a second dose of DEN at 15 weeks. A positive correlation was found between increase in the size of lesions and increased atypia in both spontaneous and DEN-induced lesions and with age for spontaneous tumors. These results support the hypothesis that mouse hepatocellular adenomas are truly neoplastic lesions in different stages of progression toward malignancy.
Naturally occurring basophilic foci (focal hepatocellular proliferative lesions) (FHPL) in the li... more Naturally occurring basophilic foci (focal hepatocellular proliferative lesions) (FHPL) in the livers of aging female F344/NCr rats could not be promoted to grow or progress into tumors after phenobarbital (PB) exposure. Instead, PB induced new unique eosinophilic hepatocellular foci and adenomas much quicker in old rats than in young rats. We now report that these foci are immunoreactiue for glutathione S-transferase n (GSTP) and that they appear to arise from some naturally occurring single and double GSTP-reactive cells and foci which occur spontaneously in the liver of aging F344 rats in an age-related fashion. PB and other nongenotoxic chemicals may act as "carcinogens" by promoting the growth (clonal expansion) of some of these putatiue spontaneously-initiated cells and foci into tumors.
Journal of Histochemistry and Cytochemistry, Feb 1, 1991
We studied the use ofacute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detectio... more We studied the use ofacute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detection of DNA-synthesizing cells in the liver and kidney ofB6C3F1 male mice. Six-week-old mice were exposed to BrdU either acutely with a single-pulse (II') injection 1 hr before sacrifice or chronically with the use of slow-release pellets or osmotic minipumps at one offour BrdU dose rates. Pellets (2.5, 10, 25, and 50 mg) and minipumps (2.5 and 10 mg equivalents) were implanted subcutaneously on the backs ofthe animals 4 or 7 days before sacrifice). BrdU incorporation into DNA was determined by immunohistochemistry using an anti-BrdU antibody. Mice chronically exposed to BrdU demonstrated increased levels of nudear labeling compared with those receiving a single-pulse injection. No time-related increases in nudear labeling were detected in hepatocytes or
Previous studies have shown that phenobarbital (PB), as well as another known liver tumor promote... more Previous studies have shown that phenobarbital (PB), as well as another known liver tumor promoter, alpha-hexachlorocyclohexane (HCH), inhibits hepatic tumor formation in infant N-nitrosodiethylamine (NDEA)-initiated C57BL/6 x C3H/He (B6C3F1) male mice. These inconsistencies in detecting PB and HCH as tumor promoters have raised important questions on the mechanism of tumor promotion in mice, as well as the reliability of the infant B6C3F1 mouse as an initiation model in two-stage carcinogenesis experiments. Therefore, in an effort to avoid the inconsistencies associated with the B6C3F1 mouse, the present study evaluated the ability of two known hepatic liver tumor promoters, di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, and phenobarbital (PB), a barbiturate, to promote hepatocellular tumorigenesis in mice of the C3H/HeNCr strain initiated during infancy. At 15 days of age, male and female C3H/HeNCr mice received either a single ip injection of NDEA (5 micrograms/g body weight) or saline. At weaning (4 weeks of age), mice were divided into 3 groups and treated with either DEHP in the diet (12,000 ppm), PB in the drinking water (500 ppm), or control drinking water and diet for 24 weeks. All mice were killed at 28 weeks of age and the number and size of hepatic foci and adenomas were evaluated. Mice exposed to NDEA+DEHP or NDEA+PB showed significant increases in the number and size of hepatic tumors compared to those receiving NDEA alone. DEHP treatment in males yielded larger adenomas than those seen in PB-treated males.(ABSTRACT TRUNCATED AT 250 WORDS)
The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of ... more The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 ± 0), 42% (1.8 ± 0.8), or 67% (2.5 ± 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 ± 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to N-nitrosodiethylamine followed by 1500 ppm DPH or 500 ppm phenobarbital, respectively. Multiplicities of hepatocellular adenomas were significantly greater than the control value in rats fed 1500 ppm DPH or 500 ppm phenobarbital (5.9 ± 4.8 and 10.1 ± 6.7, respectively), but not in the rats receiving 500 ppm DPH (2.3 ± 1.6). No rats exposed to N-nitrosodiethylamine alone or the carcinogen followed by 500 ppm DPH developed hepatocellular carcinomas, while hepatocellular carcinomas occurred in 29% or 67% of the rats given 1500 ppm DPH or 500 ppm phenobarbital, respectively, following initiation. Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylation activity in rats exposed to 500 and 1500 ppm DPH for 2 or 23 weeks were ∼50% and ∼100%, respectively, of the maximal induction caused by 500 ppm phenobarbital. Thus, in the rat model, DPH enhanced N-nitrosodiethyl-amine-initiated hepatocarcinogenesis when administered at a dose causing maximal CYP2B induction.
To investigate the promoting effects of l,4-bis[2-(3,5dichloropyridyloxy)]benzene (TCPOBOP) on li... more To investigate the promoting effects of l,4-bis[2-(3,5dichloropyridyloxy)]benzene (TCPOBOP) on liver and thyroid carcinogenesis of rats at doses that cause maximal induction of hepatic CYP2B, 5-week-old male F344 rats were given either a single i.p. dose of 75 mg N-nitrosodiethylamine (NDEA)/kg body wt in saline or saline alone. After 2 weeks the rats were fed control diet or a diet containing 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. phenobarbital (PB; a positive control group). A total of four sequential sacrifices (9, 30, 52 and 79 weeks of age) was performed. At 30 weeks the mean volume (mm 3) of hepatocellular foci in NDEA-initiated rats exposed to either dose of TCPOBOP or to PB was significantly increased as compared with rats exposed to NDEA followed by control diet (P < 0.05). In addition, the volume percentage of liver occupied by foci was significantly greater in NDEAinitiated/1000 p.p.m. TCPOBOP-promoted rats as compared with rats exposed to NDEA alone (P < 0.05, n = 6). At 52 weeks of age the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0, 83 (2.6 ± 13), 100 (3.4 ± 2.1) or 67% (2.5 ± 1.9) in rats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB respectively (n = 12). Hepatocellular carcinomas were found only in rats given 1000 p.p.m. TCPOBOP (17% incidence) or PB (8% incidence) following NDEA initiation. The incidences of thyroid follicular cell adenomas were 0, 17, 33 or 8% in rats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB respectively. Between 53 and 79 weeks of age 38% of rats treated with NDEA alone developed multiple (1.5 ± 0.8) hepatocellular adenomas. This incidence was enhanced to 100% in rats exposed to NDEA followed by either 330 or 1000 p.p.m. TCPOBOP. Multiplicities of hepatocellular adenomas were also increased significantly (
Hydrogen peroxide is a common antiseptic and disinfectant that is effective against both envelope... more Hydrogen peroxide is a common antiseptic and disinfectant that is effective against both enveloped and non-enveloped viruses, and it is sometimes used as a fumigant to achieve disinfection of indoor spaces. While it is effective as a fumigant, it cannot be used continuously, allowing for possible recontamination of the treated spaces between applications. A novel method of hydrogen peroxide application, termed "Dry Hydrogen Peroxide" (DHP™), generates molecules of hydrogen peroxide in a true gas state at concentrations low enough to be used continuously within spaces occupied by humans. This chapter explores the efficacy of DHP against a variety of viruses, both enveloped and non-enveloped. On surfaces, DHP achieved a ≥ 99.8% reduction (≥2.62 log 10 inactivation) of infectious H1N1 influenza A (enveloped) compared to the control condition within 1 hour, and it achieved a 99.8% reduction (2.62 log 10 inactivation) of infectious feline calicivirus (non-enveloped) compared to the control condition within 6 hours. DHP also achieved a 99.8% reduction 2.62 log 10 inactivation) of airborne MS2 bacteriophage (non-enveloped) within 1 hour in comparison to the control condition. These inactivation efficacy results, combined with results from recent clinical studies, indicate that DHP represents an effective adjunct technology that can mitigate viral load between intermittent applications of other types of disinfectants.
In this column, three of ABSA's experts—“The Containment Guys”—in the areas of containment eq... more In this column, three of ABSA's experts—“The Containment Guys”—in the areas of containment equipment and facilities operations answer questions about a variety of containment topics. Please e-mail questions on anything related to biocontainment facility design, operations, maintenance, and biosafety to Paul Jennette at Containment Guys@absa.org.
Waste disposal in Agricultural Animal High Containment Animal Biosafety Level 3Ag and Animal Bios... more Waste disposal in Agricultural Animal High Containment Animal Biosafety Level 3Ag and Animal Biosafety Level 4Ag (ABSL-3Ag and ABSL-4Ag) research facilities necessitates significantly more attention to detail in operations than that required in lower-containment-level laboratories. The unique features and requirements of agricultural-related research involve additional equipment and systems to safely transfer decontaminated waste out of the facility. The waste stream coming from ABSL-3Ag and ABSL-4Ag facilities, or high containment agricultural research waste, consists of many forms and differs from most research facility waste in that it is produced from research with livestock or other species loose housed, with the animal room serving as primary containment. This is in contrast to small laboratory animals being housed in primary containment caging. Waste handling equipment in agricultural research facilities may include autoclaves, eff luent decontamination systems, incinerators, high-temperature renderers, alkaline tissue digester systems, high-efficiency particulate air filtration of exhaust and supply air, gas decontamination systems, and laundry facilities. This article focuses primarily on the disposal of waste from ABSL-3Ag livestock facilities, including procedures and lessons learned over 10 years of facility operation.
Introduction: The applications of fumigation and the challenges that high-containment facilities ... more Introduction: The applications of fumigation and the challenges that high-containment facilities face in achieving effective large volume decontamination are well understood. The Biosecurity Research Institute at Kansas State University sought to evaluate a novel system within their biosafety level 3 (BSL-3) and animal biosafety level 3 agriculture (ABSL-3Ag) facility. Methods: The system chosen for this study is the CURIS â Hybrid Hydrogen Peroxide TM (HHP TM) system, comprising a mobile 36-pound (16 kg) device delivering a proprietary 7% hydrogen peroxide (H 2 O 2) solution. To examine the system's efficacy in multiple laboratory settings, two BSL-3 laboratories (2,281 [65 m 3 ] and 4,668 ft 3 [132 m 3 ]) with dropped ceiling interstitial spaces and an ABSL-3Ag necropsy suite (44,212 ft 3 [1,252 m 3 ]) with 21-foot (6.4 m) ceilings were selected. Biological indicators (BIs) of Geobacillus stearothermophilus (1.7 • 10 6 organisms) on steel spore carriers and H 2 O 2 chemical indicators (CIs) were used to provide validation. Results: After cycle optimization, the smaller laboratory had a total of 60 BIs over two treatments that demonstrated a greater than 6-log reduction of bacterial spores. The larger laboratory (192 BIs) and the necropsy suite (206 BIs) had no BIs positive for spore growth when incubated at 60°C for 24 h per manufacturer's specifications. Conclusion: Overall successful results through multiple components of this study demonstrate that the HHP device, paired with the pulsed 7% H 2 O 2 solution, achieved efficacy regardless of variables in laboratory size and layout. Perceived challenges such as 21-ft (6.4 m) ceiling heights, active equipment, and difficult to access ceiling interstitial spaces proved unfounded. Given the successful sterilization of all challenged BIs, the HHP system presents a useful alternative for high level decontamination within BSL-3 and ABSL-3Ag facilities.
The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of ... more The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 ± 0), 42% (1.8 ± 0.8), or 67% (2.5 ± 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 ± 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to...
Waste disposal in Agricultural Animal High Containment Animal Biosafety Level 3Ag and Animal Bios... more Waste disposal in Agricultural Animal High Containment Animal Biosafety Level 3Ag and Animal Biosafety Level 4Ag (ABSL-3Ag and ABSL-4Ag) research facilities necessitates significantly more attention to detail in operations than that required in lower-containment-level laboratories. The unique features and requirements of agricultural-related research involve additional equipment and systems to safely transfer decontaminated waste out of the facility. The waste stream coming from ABSL-3Ag and ABSL-4Ag facilities, or high containment agricultural research waste, consists of many forms and differs from most research facility waste in that it is produced from research with livestock or other species loose housed, with the animal room serving as primary containment. This is in contrast to small laboratory animals being housed in primary containment caging. Waste handling equipment in agricultural research facilities may include autoclaves, effluent decontamination systems, incinerators, ...
Both alveolar type II cells and Clara cells have been suggested as cells of origin of human bronc... more Both alveolar type II cells and Clara cells have been suggested as cells of origin of human bronchioloalveolar lung carcinomas and other pulmonary neoplasms, based on the presence of cell specific markers identified by immunocytochemical methods. Alveolar type II cell origin of solid and papillary lung tumors of the mouse has been demonstrated, and Clara cells have been suggested as cell of origin for hamster pulmonary neoplasms. Therefore, chemically induced bronchiolar hyperplasias and pulmonary neoplasms of Syrian golden hamsters were analyzed by avidin-biotin immunohistochemistry to localize a hamster-specific Clara cell antigen (CCA) and keratin. The hamsters had been treated subcutaneously with multiple doses of N-nitrosodiethylamine (NDEA). Proliferative lesions of low cuboidal, tall columnar, or pleomorphic cells were present within bronchioles or adjacent to airways in the alveolar parenchyma. Frequently areas of squamous cell differentiation were present focally or diffuse...
Journal of Histochemistry & Cytochemistry, 1991
We studied the use of acute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for det... more We studied the use of acute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detection of DNA-synthesizing cells in the liver and kidney of B6C3F1 male mice. Six-week-old mice were exposed to BrdU either acutely with a single-pulse (IP) injection 1 hr before sacrifice or chronically with the use of slow-release pellets or osmotic minipumps at one of four BrdU dose rates. Pellets (2.5, 10, 25, and 50 mg) and minipumps (2.5 and 10 mg equivalents) were implanted subcutaneously on the backs of the animals 4 or 7 days before sacrifice). BrdU incorporation into DNA was determined by immunohistochemistry using an anti-BrdU antibody. Mice chronically exposed to BrdU demonstrated increased levels of nuclear labeling compared with those receiving a single-pulse injection. No time-related increases in nuclear labeling were detected in hepatocytes or renal tubule cells of mice exposed to BrdU pellets and in the kidneys of mice receiving BrdU minipumps at the 7-day compared with...
Hydrogen peroxide is a common antiseptic and disinfectant that is effective against both envelope... more Hydrogen peroxide is a common antiseptic and disinfectant that is effective against both enveloped and non-enveloped viruses, and it is sometimes used as a fumigant to achieve disinfection of indoor spaces. While it is effective as a fumigant, it cannot be used continuously, allowing for possible recontamination of the treated spaces between applications. A novel method of hydrogen peroxide application, termed “Dry Hydrogen Peroxide” (DHP™), generates molecules of hydrogen peroxide in a true gas state at concentrations low enough to be used continuously within spaces occupied by humans. This chapter explores the efficacy of DHP against a variety of viruses, both enveloped and non-enveloped. On surfaces, DHP achieved a ≥ 99.8% reduction (≥2.62 log10 inactivation) of infectious H1N1 influenza A (enveloped) compared to the control condition within 1 hour, and it achieved a 99.8% reduction (2.62 log10 inactivation) of infectious feline calicivirus (non-enveloped) compared to the control...
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2002
Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drink... more Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drinking water) for up to 180 days resulted in rapid accumulation of N7- and O6-methylguanine in liver and white blood cell DNA, maximum adduct levels being reached within 1-7 days, depending on the dose. The levels of both adducts remained constant up to treatment day 28, subsequently declining slowly to about 40% of maximal levels for the liver and 60% for white blood cells by day 180. In order to elucidate the role of DNA replication in NDMA hepatocarcinogenesis, changes in liver cell labeling index (LI) were also measured on treatment days 21, 120 and 180. Although the time- and dose-dependence of the observed effects were complex, a clear trend towards increased rates of hepatocyte LI, as indicated by BrdU incorporation, with increasing NDMA doses was evident, particularly above 1 ppm, a concentration above which NDMA hepatocarcinogenicity is known to increase sharply. In contrast, no increase in Kupffer cell DNA replication was found at any of the doses employed, in accordance with the low susceptibility of these cells to NDMA-induced carcinogenesis. No significant increase in the occurrence of necrotic or apoptotic cells was noted under the treatment conditions employed. These results suggest that, in addition to the accumulation of DNA damage, alterations in hepatocyte DNA replication during the chronic NDMA exposure may influence the dose-dependence of its carcinogenic efficacy.
Journal of Toxicology and Environmental Health, 1994
The induction of immunoreactive cytochrome P-450 protein and associated catalytic activities in 1... more The induction of immunoreactive cytochrome P-450 protein and associated catalytic activities in 10-wk-old male and female Sigmodon hispidus (cotton rats) exposed for 2 wk to low dietary levels of Aroclor 1254 (0.33, 1.0, 3.3, 10, and 33 ppm), or the prototype P-450 inducers phenobarbital, DDT, clotrimazole, and beta-naphthoflavone was examined. Ethoxy-(ETR), methoxy- (MTR), pentoxy- (PTR), and benzyloxyresorufin (BZR) O-dealkylation activities were significantly increased at 0.33 ppm Aroclor for males and 1.0 ppm for females, when compared to control levels. O-Dealkylation activities peaked at 3.3 ppm for males and 10 ppm for females. ETR and MTR O-dealkylation activities were increased four- to eightfold while PTR and BZR O-dealkylation activities increased only two- to threefold. Liver/body weight ratios also increased, with the maximum ratios observed at the highest Aroclor dose, and were associated with histopathologic hepatocyte lesions. While increases in liver/body weight ratio, immunoreactive CYP2B protein, and BZR O-dealkylation were detected following phenobarbital treatment, no increase in PTR O-dealkylation activity was observed. These results demonstrate that S. hispidus (both males and females) are extremely sensitive to low dietary levels of Aroclor 1254, responding with increases in liver/body weight ratio, immunoreactive P-450 protein, and O-dealkylation activities. The cotton rat would appear to be a sensitive feral target species for detecting exposure to certain environmental contaminants.
Outbred L10 rats received 4 subcutaneous injections of 3.2 mg per rat 5-bromo-2&#39;-deoxyuri... more Outbred L10 rats received 4 subcutaneous injections of 3.2 mg per rat 5-bromo-2&#39;-deoxyuridine (BrdUrd) at 1, 3, 7 and 21 days of age. Groups of rats were sacrificed 1 h, 1 month or 49 weeks after the final injection. Tissues were fixed in 70% ethanol, embedded in paraffin and unstained sections were prepared for immunohistochemical demonstration of BrdUrd. A monoclonal antibody to BrdUrd was used with the avidin biotin peroxidase-complex (ABC) technique. The numbers of immunoreactive nuclei were tissue, cell and time-related. The labeling indices declined from 1 day to 49 weeks for all tissues studied. At 49 weeks after the last exposure to BrdUrd, many cells were still reactive, especially in tissues with normal low cell turnover (brain, uterine stroma). For cells with high turnover, including lymphocytes and ovarian germinative epithelium, few or no labeled cells remained at 49 weeks. This study provides clear evidence for the persistence of BrdUrd in normal tissues, some of which may be targets for the carcinogenic effect of the chemical and others which are not targets.
The progression of hepatocellular adenomas to carcinomas has been less well documented in mice th... more The progression of hepatocellular adenomas to carcinomas has been less well documented in mice than in rats. We studied progression of spontaneous and chemically induced hepatocellular adenomas in male C3H/HeNCr mice by image analysis. Spontaneous lesions in 15, 18 and 21 month old untreated male C3H/HeNCr mice and experimentally induced lesions were examined. Experimental group 1 received a single i.p. injection of N-nitrosodiethylamine (DEN) (5 mg/kg body wt) at 15 days of age. Groups 2 and 3 were injected a second time with DEN at 15 or 20 weeks of age (75 mg/kg body wt), with interim sacrifices at 11, 16 and 34 weeks after the second DEN injection. Atypia in adenomas were classified into four grades according to cell size, tinctorial changes, cellular pleomorphism and trabecular pattern. At earlier stages of the neoplastic process (11 or 16 weeks after the second DEN dose), most adenomas were well-differentiated lesions with no atypia or focal grade 1 or 2 atypia. At later stages (34 weeks after the second DEN dose), a large proportion of hepatocellular tumors were classified as adenoma with grade 3 atypia or carcinoma. The proportion of carcinomas in mice treated with a second dose of DEN at 20 weeks of age was significantly higher than in mice treated with a single dose of DEN or in mice given a second dose of DEN at 15 weeks. A positive correlation was found between increase in the size of lesions and increased atypia in both spontaneous and DEN-induced lesions and with age for spontaneous tumors. These results support the hypothesis that mouse hepatocellular adenomas are truly neoplastic lesions in different stages of progression toward malignancy.
Naturally occurring basophilic foci (focal hepatocellular proliferative lesions) (FHPL) in the li... more Naturally occurring basophilic foci (focal hepatocellular proliferative lesions) (FHPL) in the livers of aging female F344/NCr rats could not be promoted to grow or progress into tumors after phenobarbital (PB) exposure. Instead, PB induced new unique eosinophilic hepatocellular foci and adenomas much quicker in old rats than in young rats. We now report that these foci are immunoreactiue for glutathione S-transferase n (GSTP) and that they appear to arise from some naturally occurring single and double GSTP-reactive cells and foci which occur spontaneously in the liver of aging F344 rats in an age-related fashion. PB and other nongenotoxic chemicals may act as "carcinogens" by promoting the growth (clonal expansion) of some of these putatiue spontaneously-initiated cells and foci into tumors.
Journal of Histochemistry and Cytochemistry, Feb 1, 1991
We studied the use ofacute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detectio... more We studied the use ofacute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detection of DNA-synthesizing cells in the liver and kidney ofB6C3F1 male mice. Six-week-old mice were exposed to BrdU either acutely with a single-pulse (II') injection 1 hr before sacrifice or chronically with the use of slow-release pellets or osmotic minipumps at one offour BrdU dose rates. Pellets (2.5, 10, 25, and 50 mg) and minipumps (2.5 and 10 mg equivalents) were implanted subcutaneously on the backs ofthe animals 4 or 7 days before sacrifice). BrdU incorporation into DNA was determined by immunohistochemistry using an anti-BrdU antibody. Mice chronically exposed to BrdU demonstrated increased levels of nudear labeling compared with those receiving a single-pulse injection. No time-related increases in nudear labeling were detected in hepatocytes or
Previous studies have shown that phenobarbital (PB), as well as another known liver tumor promote... more Previous studies have shown that phenobarbital (PB), as well as another known liver tumor promoter, alpha-hexachlorocyclohexane (HCH), inhibits hepatic tumor formation in infant N-nitrosodiethylamine (NDEA)-initiated C57BL/6 x C3H/He (B6C3F1) male mice. These inconsistencies in detecting PB and HCH as tumor promoters have raised important questions on the mechanism of tumor promotion in mice, as well as the reliability of the infant B6C3F1 mouse as an initiation model in two-stage carcinogenesis experiments. Therefore, in an effort to avoid the inconsistencies associated with the B6C3F1 mouse, the present study evaluated the ability of two known hepatic liver tumor promoters, di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, and phenobarbital (PB), a barbiturate, to promote hepatocellular tumorigenesis in mice of the C3H/HeNCr strain initiated during infancy. At 15 days of age, male and female C3H/HeNCr mice received either a single ip injection of NDEA (5 micrograms/g body weight) or saline. At weaning (4 weeks of age), mice were divided into 3 groups and treated with either DEHP in the diet (12,000 ppm), PB in the drinking water (500 ppm), or control drinking water and diet for 24 weeks. All mice were killed at 28 weeks of age and the number and size of hepatic foci and adenomas were evaluated. Mice exposed to NDEA+DEHP or NDEA+PB showed significant increases in the number and size of hepatic tumors compared to those receiving NDEA alone. DEHP treatment in males yielded larger adenomas than those seen in PB-treated males.(ABSTRACT TRUNCATED AT 250 WORDS)
The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of ... more The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 ± 0), 42% (1.8 ± 0.8), or 67% (2.5 ± 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 ± 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to N-nitrosodiethylamine followed by 1500 ppm DPH or 500 ppm phenobarbital, respectively. Multiplicities of hepatocellular adenomas were significantly greater than the control value in rats fed 1500 ppm DPH or 500 ppm phenobarbital (5.9 ± 4.8 and 10.1 ± 6.7, respectively), but not in the rats receiving 500 ppm DPH (2.3 ± 1.6). No rats exposed to N-nitrosodiethylamine alone or the carcinogen followed by 500 ppm DPH developed hepatocellular carcinomas, while hepatocellular carcinomas occurred in 29% or 67% of the rats given 1500 ppm DPH or 500 ppm phenobarbital, respectively, following initiation. Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylation activity in rats exposed to 500 and 1500 ppm DPH for 2 or 23 weeks were ∼50% and ∼100%, respectively, of the maximal induction caused by 500 ppm phenobarbital. Thus, in the rat model, DPH enhanced N-nitrosodiethyl-amine-initiated hepatocarcinogenesis when administered at a dose causing maximal CYP2B induction.
To investigate the promoting effects of l,4-bis[2-(3,5dichloropyridyloxy)]benzene (TCPOBOP) on li... more To investigate the promoting effects of l,4-bis[2-(3,5dichloropyridyloxy)]benzene (TCPOBOP) on liver and thyroid carcinogenesis of rats at doses that cause maximal induction of hepatic CYP2B, 5-week-old male F344 rats were given either a single i.p. dose of 75 mg N-nitrosodiethylamine (NDEA)/kg body wt in saline or saline alone. After 2 weeks the rats were fed control diet or a diet containing 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. phenobarbital (PB; a positive control group). A total of four sequential sacrifices (9, 30, 52 and 79 weeks of age) was performed. At 30 weeks the mean volume (mm 3) of hepatocellular foci in NDEA-initiated rats exposed to either dose of TCPOBOP or to PB was significantly increased as compared with rats exposed to NDEA followed by control diet (P < 0.05). In addition, the volume percentage of liver occupied by foci was significantly greater in NDEAinitiated/1000 p.p.m. TCPOBOP-promoted rats as compared with rats exposed to NDEA alone (P < 0.05, n = 6). At 52 weeks of age the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0, 83 (2.6 ± 13), 100 (3.4 ± 2.1) or 67% (2.5 ± 1.9) in rats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB respectively (n = 12). Hepatocellular carcinomas were found only in rats given 1000 p.p.m. TCPOBOP (17% incidence) or PB (8% incidence) following NDEA initiation. The incidences of thyroid follicular cell adenomas were 0, 17, 33 or 8% in rats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB respectively. Between 53 and 79 weeks of age 38% of rats treated with NDEA alone developed multiple (1.5 ± 0.8) hepatocellular adenomas. This incidence was enhanced to 100% in rats exposed to NDEA followed by either 330 or 1000 p.p.m. TCPOBOP. Multiplicities of hepatocellular adenomas were also increased significantly (
Hydrogen peroxide is a common antiseptic and disinfectant that is effective against both envelope... more Hydrogen peroxide is a common antiseptic and disinfectant that is effective against both enveloped and non-enveloped viruses, and it is sometimes used as a fumigant to achieve disinfection of indoor spaces. While it is effective as a fumigant, it cannot be used continuously, allowing for possible recontamination of the treated spaces between applications. A novel method of hydrogen peroxide application, termed "Dry Hydrogen Peroxide" (DHP™), generates molecules of hydrogen peroxide in a true gas state at concentrations low enough to be used continuously within spaces occupied by humans. This chapter explores the efficacy of DHP against a variety of viruses, both enveloped and non-enveloped. On surfaces, DHP achieved a ≥ 99.8% reduction (≥2.62 log 10 inactivation) of infectious H1N1 influenza A (enveloped) compared to the control condition within 1 hour, and it achieved a 99.8% reduction (2.62 log 10 inactivation) of infectious feline calicivirus (non-enveloped) compared to the control condition within 6 hours. DHP also achieved a 99.8% reduction 2.62 log 10 inactivation) of airborne MS2 bacteriophage (non-enveloped) within 1 hour in comparison to the control condition. These inactivation efficacy results, combined with results from recent clinical studies, indicate that DHP represents an effective adjunct technology that can mitigate viral load between intermittent applications of other types of disinfectants.
In this column, three of ABSA's experts—“The Containment Guys”—in the areas of containment eq... more In this column, three of ABSA's experts—“The Containment Guys”—in the areas of containment equipment and facilities operations answer questions about a variety of containment topics. Please e-mail questions on anything related to biocontainment facility design, operations, maintenance, and biosafety to Paul Jennette at Containment Guys@absa.org.
Waste disposal in Agricultural Animal High Containment Animal Biosafety Level 3Ag and Animal Bios... more Waste disposal in Agricultural Animal High Containment Animal Biosafety Level 3Ag and Animal Biosafety Level 4Ag (ABSL-3Ag and ABSL-4Ag) research facilities necessitates significantly more attention to detail in operations than that required in lower-containment-level laboratories. The unique features and requirements of agricultural-related research involve additional equipment and systems to safely transfer decontaminated waste out of the facility. The waste stream coming from ABSL-3Ag and ABSL-4Ag facilities, or high containment agricultural research waste, consists of many forms and differs from most research facility waste in that it is produced from research with livestock or other species loose housed, with the animal room serving as primary containment. This is in contrast to small laboratory animals being housed in primary containment caging. Waste handling equipment in agricultural research facilities may include autoclaves, eff luent decontamination systems, incinerators, high-temperature renderers, alkaline tissue digester systems, high-efficiency particulate air filtration of exhaust and supply air, gas decontamination systems, and laundry facilities. This article focuses primarily on the disposal of waste from ABSL-3Ag livestock facilities, including procedures and lessons learned over 10 years of facility operation.
Introduction: The applications of fumigation and the challenges that high-containment facilities ... more Introduction: The applications of fumigation and the challenges that high-containment facilities face in achieving effective large volume decontamination are well understood. The Biosecurity Research Institute at Kansas State University sought to evaluate a novel system within their biosafety level 3 (BSL-3) and animal biosafety level 3 agriculture (ABSL-3Ag) facility. Methods: The system chosen for this study is the CURIS â Hybrid Hydrogen Peroxide TM (HHP TM) system, comprising a mobile 36-pound (16 kg) device delivering a proprietary 7% hydrogen peroxide (H 2 O 2) solution. To examine the system's efficacy in multiple laboratory settings, two BSL-3 laboratories (2,281 [65 m 3 ] and 4,668 ft 3 [132 m 3 ]) with dropped ceiling interstitial spaces and an ABSL-3Ag necropsy suite (44,212 ft 3 [1,252 m 3 ]) with 21-foot (6.4 m) ceilings were selected. Biological indicators (BIs) of Geobacillus stearothermophilus (1.7 • 10 6 organisms) on steel spore carriers and H 2 O 2 chemical indicators (CIs) were used to provide validation. Results: After cycle optimization, the smaller laboratory had a total of 60 BIs over two treatments that demonstrated a greater than 6-log reduction of bacterial spores. The larger laboratory (192 BIs) and the necropsy suite (206 BIs) had no BIs positive for spore growth when incubated at 60°C for 24 h per manufacturer's specifications. Conclusion: Overall successful results through multiple components of this study demonstrate that the HHP device, paired with the pulsed 7% H 2 O 2 solution, achieved efficacy regardless of variables in laboratory size and layout. Perceived challenges such as 21-ft (6.4 m) ceiling heights, active equipment, and difficult to access ceiling interstitial spaces proved unfounded. Given the successful sterilization of all challenged BIs, the HHP system presents a useful alternative for high level decontamination within BSL-3 and ABSL-3Ag facilities.
The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of ... more The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 ± 0), 42% (1.8 ± 0.8), or 67% (2.5 ± 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 ± 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to...
Waste disposal in Agricultural Animal High Containment Animal Biosafety Level 3Ag and Animal Bios... more Waste disposal in Agricultural Animal High Containment Animal Biosafety Level 3Ag and Animal Biosafety Level 4Ag (ABSL-3Ag and ABSL-4Ag) research facilities necessitates significantly more attention to detail in operations than that required in lower-containment-level laboratories. The unique features and requirements of agricultural-related research involve additional equipment and systems to safely transfer decontaminated waste out of the facility. The waste stream coming from ABSL-3Ag and ABSL-4Ag facilities, or high containment agricultural research waste, consists of many forms and differs from most research facility waste in that it is produced from research with livestock or other species loose housed, with the animal room serving as primary containment. This is in contrast to small laboratory animals being housed in primary containment caging. Waste handling equipment in agricultural research facilities may include autoclaves, effluent decontamination systems, incinerators, ...
Both alveolar type II cells and Clara cells have been suggested as cells of origin of human bronc... more Both alveolar type II cells and Clara cells have been suggested as cells of origin of human bronchioloalveolar lung carcinomas and other pulmonary neoplasms, based on the presence of cell specific markers identified by immunocytochemical methods. Alveolar type II cell origin of solid and papillary lung tumors of the mouse has been demonstrated, and Clara cells have been suggested as cell of origin for hamster pulmonary neoplasms. Therefore, chemically induced bronchiolar hyperplasias and pulmonary neoplasms of Syrian golden hamsters were analyzed by avidin-biotin immunohistochemistry to localize a hamster-specific Clara cell antigen (CCA) and keratin. The hamsters had been treated subcutaneously with multiple doses of N-nitrosodiethylamine (NDEA). Proliferative lesions of low cuboidal, tall columnar, or pleomorphic cells were present within bronchioles or adjacent to airways in the alveolar parenchyma. Frequently areas of squamous cell differentiation were present focally or diffuse...
Journal of Histochemistry & Cytochemistry, 1991
We studied the use of acute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for det... more We studied the use of acute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detection of DNA-synthesizing cells in the liver and kidney of B6C3F1 male mice. Six-week-old mice were exposed to BrdU either acutely with a single-pulse (IP) injection 1 hr before sacrifice or chronically with the use of slow-release pellets or osmotic minipumps at one of four BrdU dose rates. Pellets (2.5, 10, 25, and 50 mg) and minipumps (2.5 and 10 mg equivalents) were implanted subcutaneously on the backs of the animals 4 or 7 days before sacrifice). BrdU incorporation into DNA was determined by immunohistochemistry using an anti-BrdU antibody. Mice chronically exposed to BrdU demonstrated increased levels of nuclear labeling compared with those receiving a single-pulse injection. No time-related increases in nuclear labeling were detected in hepatocytes or renal tubule cells of mice exposed to BrdU pellets and in the kidneys of mice receiving BrdU minipumps at the 7-day compared with...
Hydrogen peroxide is a common antiseptic and disinfectant that is effective against both envelope... more Hydrogen peroxide is a common antiseptic and disinfectant that is effective against both enveloped and non-enveloped viruses, and it is sometimes used as a fumigant to achieve disinfection of indoor spaces. While it is effective as a fumigant, it cannot be used continuously, allowing for possible recontamination of the treated spaces between applications. A novel method of hydrogen peroxide application, termed “Dry Hydrogen Peroxide” (DHP™), generates molecules of hydrogen peroxide in a true gas state at concentrations low enough to be used continuously within spaces occupied by humans. This chapter explores the efficacy of DHP against a variety of viruses, both enveloped and non-enveloped. On surfaces, DHP achieved a ≥ 99.8% reduction (≥2.62 log10 inactivation) of infectious H1N1 influenza A (enveloped) compared to the control condition within 1 hour, and it achieved a 99.8% reduction (2.62 log10 inactivation) of infectious feline calicivirus (non-enveloped) compared to the control...
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2002
Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drink... more Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drinking water) for up to 180 days resulted in rapid accumulation of N7- and O6-methylguanine in liver and white blood cell DNA, maximum adduct levels being reached within 1-7 days, depending on the dose. The levels of both adducts remained constant up to treatment day 28, subsequently declining slowly to about 40% of maximal levels for the liver and 60% for white blood cells by day 180. In order to elucidate the role of DNA replication in NDMA hepatocarcinogenesis, changes in liver cell labeling index (LI) were also measured on treatment days 21, 120 and 180. Although the time- and dose-dependence of the observed effects were complex, a clear trend towards increased rates of hepatocyte LI, as indicated by BrdU incorporation, with increasing NDMA doses was evident, particularly above 1 ppm, a concentration above which NDMA hepatocarcinogenicity is known to increase sharply. In contrast, no increase in Kupffer cell DNA replication was found at any of the doses employed, in accordance with the low susceptibility of these cells to NDMA-induced carcinogenesis. No significant increase in the occurrence of necrotic or apoptotic cells was noted under the treatment conditions employed. These results suggest that, in addition to the accumulation of DNA damage, alterations in hepatocyte DNA replication during the chronic NDMA exposure may influence the dose-dependence of its carcinogenic efficacy.
Journal of Toxicology and Environmental Health, 1994
The induction of immunoreactive cytochrome P-450 protein and associated catalytic activities in 1... more The induction of immunoreactive cytochrome P-450 protein and associated catalytic activities in 10-wk-old male and female Sigmodon hispidus (cotton rats) exposed for 2 wk to low dietary levels of Aroclor 1254 (0.33, 1.0, 3.3, 10, and 33 ppm), or the prototype P-450 inducers phenobarbital, DDT, clotrimazole, and beta-naphthoflavone was examined. Ethoxy-(ETR), methoxy- (MTR), pentoxy- (PTR), and benzyloxyresorufin (BZR) O-dealkylation activities were significantly increased at 0.33 ppm Aroclor for males and 1.0 ppm for females, when compared to control levels. O-Dealkylation activities peaked at 3.3 ppm for males and 10 ppm for females. ETR and MTR O-dealkylation activities were increased four- to eightfold while PTR and BZR O-dealkylation activities increased only two- to threefold. Liver/body weight ratios also increased, with the maximum ratios observed at the highest Aroclor dose, and were associated with histopathologic hepatocyte lesions. While increases in liver/body weight ratio, immunoreactive CYP2B protein, and BZR O-dealkylation were detected following phenobarbital treatment, no increase in PTR O-dealkylation activity was observed. These results demonstrate that S. hispidus (both males and females) are extremely sensitive to low dietary levels of Aroclor 1254, responding with increases in liver/body weight ratio, immunoreactive P-450 protein, and O-dealkylation activities. The cotton rat would appear to be a sensitive feral target species for detecting exposure to certain environmental contaminants.
Outbred L10 rats received 4 subcutaneous injections of 3.2 mg per rat 5-bromo-2&#39;-deoxyuri... more Outbred L10 rats received 4 subcutaneous injections of 3.2 mg per rat 5-bromo-2&#39;-deoxyuridine (BrdUrd) at 1, 3, 7 and 21 days of age. Groups of rats were sacrificed 1 h, 1 month or 49 weeks after the final injection. Tissues were fixed in 70% ethanol, embedded in paraffin and unstained sections were prepared for immunohistochemical demonstration of BrdUrd. A monoclonal antibody to BrdUrd was used with the avidin biotin peroxidase-complex (ABC) technique. The numbers of immunoreactive nuclei were tissue, cell and time-related. The labeling indices declined from 1 day to 49 weeks for all tissues studied. At 49 weeks after the last exposure to BrdUrd, many cells were still reactive, especially in tissues with normal low cell turnover (brain, uterine stroma). For cells with high turnover, including lymphocytes and ovarian germinative epithelium, few or no labeled cells remained at 49 weeks. This study provides clear evidence for the persistence of BrdUrd in normal tissues, some of which may be targets for the carcinogenic effect of the chemical and others which are not targets.
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Papers by John Henneman