Detection of a cellular oncogene in spontaneous liver tumors of B6C3F1 mice.

An active cellular oncogene was demonstrated in hepatocellular neoplasms arising spontaneously in 24-month-old B6C3F1 mice. DNA isolated from the tumorous tissue and transfected into NIH 3T3 cells showed an 82 percent (9 of 11 animals) frequency of foci induction. In contrast, DNA isolated from the surrounding nontumorous hepatic tissue from the same animals and DNA from other 24-month-old B6C3F1 mice without tumors did not cause transformation in the NIH 3T3 cell assay. This strain of mouse is used extensively in carcinogen bioassays, and the observed high frequency of transformation (82 percent, compared to 10 to 20 percent in humans) supports the concept that the B6C3F1 mouse is hypersusceptible to liver tumor development. It also emphasizes the need to further understand the mechanisms of oncogene activation in animals used for long-term studies of toxicity and oncogenicity before evaluating potential human risk.

Streamside management zones effectiveness for protecting water quality after forestland application of biosolids.

Biosolids, materials resulting from domestic sewage treatment, are surface applied to forest soils to increase phosphorus (P), nitrate, and ammonium availability. Retaining streamside management zones (SMZs) can limit nutrient pollution of streams. We delineated 15-m SMZs along three intermittent streams in an 18-yr-old Pinus taeda L. plantation. We applied biosolids at a rate of 1120 and 629 kg ha(-1) of total nitrogen and total P outside the SMZ on one side of each of the streams while maintaining the other side of the stream as control. We collected water samples from the three treated and six reference streams and from the perennial stream upstream and downstream from the intermittent streams for 12 mo after treatment. Along transects perpendicular to the treated streams, we collected overland flow samples, soil solution samples at 60 cm, and extracts from ion exchange membranes (IEMs) placed in the surface soil. We observed significantly elevated P concentrations adjacent to the stream in overland flow during one period on the treated side of the stream. We found significantly elevated nitrate concentrations outside the SMZ in the treated-side soil solution samples, in which concentrations remained below 1.5 mg L(-1). Phosphorus, nitrate, and ammonium concentrations outside the SMZ in treated-side IEM extracts showed significant increases after biosolids application, returning to near control levels after 1 yr. Phosphorus, nitrate, and ammonium concentrations in IEM extracts were not different adjacent to the streams. Stream P, nitrate, and ammonium concentrations showed few differences downstream from the treatment with concentrations below 1.5 mg L(-1). Our results indicate that at 15 m, SMZ protected streams from P, nitrate, and ammonium pollution for the first year after biosolids application to adjacent loblolly pine plantations in the Virginia Piedmont.

Post-fertilization physiology and growth performance of loblolly pine clones.

The physiological processes leading to enhanced growth of loblolly pine (Pinus taeda L.) following fertilization are not clearly understood. Part of the debate revolves around the temporal response of net photosynthetic rate (A(n)) to fertilization and whether the A(n) response is always positive. We measured light-saturated photosynthetic rate (A(sat)), dark respiration rate, growth and crown silhouette area in eight clones of loblolly pine before and after nitrogen (N) fertilization (112 kg ha(-1)) to track the initial physiological changes prior to any changes in growth. Overall, there were positive photosynthetic and growth responses to fertilization; however, there were pronounced physiological and growth differences among clones, even among clones with the same parents. Clones 4, 6 and 7 showed large volume growth and A(sat) responses to fertilization. Clone 1 and Clone 8 (a full-sibling of Clone 7) mainly showed a volume growth response, whereas Clone 2 (full-sibling of Clone 1) showed an A(sat) response only. Clone 5 (full-sibling of Clone 6) showed little response to fertilization, whereas Clone 3 (full-sibling of Clone 4) showed a negative A(sat) response. Thus, within-family variation warrants further study to ensure that relatively expensive clonal material is used efficiently.

Roles of 2-haloethylene oxides and 2-haloacetaldehydes derived from vinyl bromide and vinyl chloride in irreversible binding to protein and DNA.

The metabolism of [1,2-14C]vinyl bromide (VBR) to products irreversibly bound to DNA and protein was examined in rat liver microsomes, reconstituted cytochrome P-450 systems, and isolated hepatocytes. A role for cytochrome P-450 was confirmed using inhibition and reconstitution experiments. The major form of cytochrome P-450 involved in VBR metabolism does not appear to be either of the major isozymes induced by phenobarbital or beta-naphthoflavone, as determined by induction, reconstitution, and antibody inhibition studies. 2-Bromoethylene oxide and 2-bromoacetaldehyde, suspected metabolites of VBR, were synthesized and found to be substrates for rat liver epoxide hydrolase and equine liver alcohol dehydrogenase, respectively. These enzymes were used to probe the roles of the two possible metabolites in the irreversible binding of products of VBR to protein and DNA. Alcohol dehydrogenase was more effective than epoxide hydrolase in inhibiting the binding of VBR metabolites to protein in microsomal incubations. Epoxide hydrolase was effective in inhibiting the binding of VBR or vinyl chloride metabolites to calf thymus DNA added to such systems, but alcohol dehydrogenase was not. Similar results were obtained for binding of VBR metabolites to DNA in a reconstituted enzyme system. Reduced glutathione blocked nonenzymatic binding of 2-bromo[1,2-14C]acetaldehyde to protein but not DNA. Binding of vinyl chloride and VBR metabolites to protein was blocked by reduced glutathione, but binding to DNA was not. These results are consistent with the view that 2-haloethylene oxides are the major alkylating agents bound to DNA, and 2-haloacetaldehydes are the major alkylating agents bound to protein in these experimental systems. Studies with labeled 2-bromoacetaldehyde indicate that the slow kinetics of DNA binding by this compound is responsible in part for this phenomenon. Studies with isolated rat hepatocytes suggest that a significant portion of the total and reactive metabolites are able to leave these cells. In these systems, binding of metabolites of vinyl chloride to DNA outside the hepatocytes could be partially blocked by epoxide hydrolase or by alcohol dehydrogenase, implying that, as target farther away from sources of reactive species are considered, the stabilities of these species become more important for reaction with nucleophilic sites.

Gene expression and cell proliferation in rat liver after 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure.

Recently 4 genes (plasminogen activator inhibitor 2, interleukin 1 beta, clone 1, and clone 141) that are transcriptionally or posttranscriptionally responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been cloned from a human skin keratinocyte cell line. We determined whether these genes were expressed in the livers of Sprague-Dawley rats following exposure to TCDD and whether there was a relationship between expression and hepatic cell proliferation. TCDD was administered by using a dose loading/maintenance regimen to achieve rapid quasi-steady-state TCDD liver concentrations of 0.03, 30, or 150 ng/g of liver. Gene expression was determined by Northern analysis using polyadenylated mRNA isolated from liver tissue of male and female animals exposed to TCDD for 1 or 14 days and hybridized with the human complementary DNA clone corresponding to one of the four human TCDD-responsive genes. Under low-stringency hybridization conditions, only the expression of clone 1 could be detected. A dose- and time-dependent expression of this gene was observed in the liver of both male and female rats. Expression of clone 1 was not detected in rats subjected to either a two-thirds partial hepatectomy or exposure to a single administration of the hepatic tumor promoters Wy-14643, carbon tetrachloride, or phenobarbital at doses that induce hepatic cell proliferation. Liver:body weight ratios were elevated in rats exposed to the middle and high TCDD doses. Histopathological observation and analysis of serum enzyme levels indicated no evidence of TCDD-induced liver necrosis. Cell proliferation was evaluated immunohistochemically after 7-day 5-bromo-2'-deoxyuridine administration. No increase in total hepatic labeling index was observed for any of the TCDD-exposed treatment groups compared to controls at week 1 or week 2. An increase in the periportal hepatocyte proliferation labeling pattern was observed in TCDD-treated animals. While these results demonstrate that a human TCDD-responsive gene is expressed in the liver of TCDD-treated male and female Sprague-Dawley rats, the expression of this gene is not linked to hepatic cell proliferation or the sex-specific tumor-promoting activity of TCDD.

Mutational analysis of the H-ras oncogene in spontaneous C57BL/6 x C3H/He mouse liver tumors and tumors induced with genotoxic and nongenotoxic hepatocarcinogens.

The frequency and mutational profile of H-ras gene activation were determined in spontaneous liver tumors of male C57BL/6 x C3H/He mice and in tumors induced with the genotoxic hepatocarcinogen benzidine.2 HCl or the nongenotoxic hepatocarcinogens phenobarbital, chloroform, and ciprofibrate. DNA sequence analysis of the H-ras gene from representative tumors revealed that 32 of 50 (64%) spontaneous tumors and 13 of 22 (59%) benzidine.2 HCl-induced tumors contained a point mutation in codon 61. Tumors induced with the nongenotoxic agents had a much lower frequency of codon 61 mutations, i.e., phenobarbital, 1 of 15 (7%); chloroform, 5 of 24 (21%), and ciprofibrate, 8 of 39 (21%). No mutations were observed at codons 12, 13, and 117 in tumors from any of the groups. Only three base pair substitutions within codon 61 were found. The one most frequently detected in all of the groups was a C.G to A.T transversion at the first nucleotide position, occurring at a 59%, 85%, 100%, 80%, and 88% frequency in the spontaneous tumors and in the tumors induced with benzidine 2.Hcl, phenobarbital, chloroform, and ciprofibrate, respectively. In these same groups an A.T to G.C transition or an A.T to T.A transversion at the second nucleotide position occurred at a frequency of 34%, 8%, 0%, 0%, and 12%, and 6%, 8%, 0%, 20%, and 0%, respectively. The number of tumors carrying an activated H-ras gene in the nongenotoxic treatment groups is within the range that would be expected if those animals had not received any treatment. This indicates that the activation of the H-ras gene in those tumors is probably the result of a spontaneous event. The data suggest that these toxicologically and pharmacologically diverse nongenotoxic hepatocarcinogens increase the frequency of liver tumors but do not induce mutations in the H-ras gene. Instead these agents appear to interact with a population of cells that do not contain an activated H-ras gene. This suggests that the mechanisms of tumor development by these nongenotoxic carcinogens differ at least partially from the mechanisms responsible for the development of spontaneous tumors or those induced by a typical genotoxic agent.

Inhibitory effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on rat hepatocyte proliferation induced by 2/3 partial hepatectomy.

To better understand the mode of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced alterations in hepatic cell proliferation and the potential link to tumour-promoting activity, we investigated the effects of TCDD on the expression of certain key genes involved in liver cell growth and the effects of TCDD on induced hepatocyte cell proliferation. Gene expression analysis was conducted, by Northern blot hybridization, using RNA isolated from female Sprague-Dawley rat livers collected at various times during a 14-day dosing period with TCDD known to produce alterations in cell proliferation. No major changes were observed in the expression of the transforming growth factors TGF-alpha and TGF-beta or in oncogenes ras, src and myc. However, the expression of the transcription factors C/EBP, HNF-1 alpha and HNF-4 decreased after 14 days of TCDD treatment. To investigate how TCDD affects hepatic growth, cell proliferation analysis was conducted in rats stimulated to undergo hepatocyte proliferation following either 2/3 partial hepatectomy or lead nitrate treatment. Cell proliferation was quantified by means of immunocytochemical detection of Proliferating Cell Nuclear Antigen (PCNA). Fourteen days of pretreatment with TCDD caused an overall inhibition of hepatocytes in the growth fraction (G1, S, G2 and M) from 61 +/- 3% in the control-partial hepatectomy group to 41 +/- 3% in the TCDD-partial hepatectomy group. A periportal pattern of cell proliferation was observed in the TCDD-partial hepatectomy group as compared to the panlobular pattern of cell proliferation in the control-partial hepatectomy group. TCDD pretreatment also produced an inhibition of cell proliferation induced by the liver mitogen lead nitrate. TCDD-induced inhibition of hepatocyte proliferation could play a role in TCDD tumour promotion and hepatocarcinogenesis through the creation of a environment whereby preneoplastic cells continue to expand while normal hepatocyte proliferation is inhibited.

Adverse effects of outpatient parenteral antibiotic therapy.

PURPOSE: Although home parenteral antimicrobial therapy has become common, few studies have carefully examined its adverse effects. SUBJECTS AND METHODS: We retrospectively reviewed the medical records of 269 patients who received 291 courses of home parenteral antimicrobial therapy through a hospital-based home infusion program during a 2-year period. Patients with human immunodeficiency virus (HIV) infection were not included. RESULTS: The majority (59%) of patients were treated for bone and joint infections. Patients had a mean age of 47 years. The mean duration of antibiotic therapy was 40 days. Of monitored courses, leukopenia occurred in 16%, neutropenia in 7%, thrombocytopenia in 4%, and eosinophilia in 12%, usually after a month of therapy; these adverse effects were most frequently associated with the use of beta-lactam antibiotics. Nephrotoxicity occurred in 8% of monitored courses at a mean of 27 days and was most commonly associated with amphotericin B. Diarrhea occurred in 7% and rash in 4% of patients, and both were most commonly seen with beta-lactam antibiotics. Of those patients with permanent indwelling catheters, 11% of those with central catheters and 9% of those with peripherally inserted central catheters (PICCs) developed line complications. Overall, 8% of patients required rehospitalization. CONCLUSION: Home infusion antibiotic therapy exposes patients to the complications associated with inpatient antibiotic therapy and needs to be monitored closely to prevent serious complications and rehospitalizations.

Mechanistic considerations for carcinogenic risk estimation: chloroform.

Chloroform has been reported to induce cancer in rodents after chronic administration of high doses by gavage. However, the interpretation of these findings is hampered by a lack of knowledge concerning the relative roles of genetic and nongenetic mechanisms in these bioassays. The present studies were carried out in male B6C3F1 mice in order to investigate the potential of chloroform to induce genetic damage and/or organ toxicity at the sites where tumors have been observed in the various bioassays. These studies revealed that carcinogenic doses of chloroform produced severe necrosis at the sites where tumors later developed. This was demonstrated by light microscopy as well as by determination of the cellular regeneration index following administration of 3H-thymidine. Noncarcinogenic doses of chloroform failed to induce these responses. In contrast, studies of DNA alkylation and DNA repair in vivo failed to give any indication that chloroform had produced the type of genetic alterations associated with known genotoxic chemicals. These data suggest that the primary mechanism of chloroform-induced carcinogenesis is nongenetic in nature. If the same mechanism predominates in man, there should be little to no carcinogenic risk associated with exposure to noncytotoxic levels of chloroform.

Research strategy in industrial toxicology.

While much of industrial toxicology is observational in character, pursuit of specific research is needed to facilitate the overall evaluation of potential toxicity for man. Two such areas are the application of physiologic pharmacokinetic models to inter-species extrapolation of toxic effects and an understanding of the role of cellular oncogenes in the process of spontaneous tumor formation in animals. A physiologic pharmacokinetic model was developed for methylene chloride (MeCl2) which describes the fate of MeCl2 and its metabolic products in numerous species including the mouse, rat, hamster and man. This model has been used to predict specific tissue concentrations of critical metabolic reaction products in target tissues between animals and man. If it is assumed that toxicity is related to target tissue concentrations such methodology provides a means of relating interspecies toxicity to absorbed dose. This methodology precludes the necessity of using arbitrary factors in relating animal toxicity data to man. A particular controversial issue in animal toxicology is the significance of the enhancement of animal tumors in tissues which already have a high spontaneous incidence. Without a better understanding of the basic process of spontaneous tumor formation it remains difficult to interpret results from chemical treatment. In particular spontaneous liver tumors in the B6C3F1 mouse have been shown to contain an activated cellular oncogene identified as H-RAS. The activated cellular oncogene is present in tumor tissue only and not in surrounding normal liver tissue. Of particular significance is the high frequency of activation in these mouse liver tumors (82%) compared to a 10-20% incidence of oncogenes present in a variety of human tumors. This suggests the ultra sensitivity of this mouse strain to liver tumor induction. Additional studies in progress are designed to determine whether genotoxic and nongenotoxic hepatocarcinogens show differences in oncogene activation.

Activation of a cellular proto-oncogene in spontaneous liver tumor tissue of the B6C3F1 mouse.

The controversy surrounding the interpretation of observed increases in the high spontaneous liver tumor incidence of the B6C3F1 mouse after administration of certain chemical agents necessitates a mechanistic understanding into the nature of tumor development in this particular strain of mouse. Recently, cancer genes (oncogenes) have been detected in the DNA from a variety of human tumors and tumor cell lines. These genes have been implicated to play a role in the transformation of normal cells into cancerous ones. To investigate the role that cellular oncogenes might play in the development of spontaneous liver tumors in the B6C3F1 mouse, DNA was isolated from spontaneously occurring liver tumors and transfected into NIH 3T3 fibroblasts. DNA from this tumor tissue was capable of transforming NIH 3T3 cells from 82% of the animals examined strongly suggesting the presence of an active cellular oncogene. In contrast, DNA isolated from surrounding non-tumorous liver tissue and liver tissue from non-tumor bearing mice did not cause any transformation in the NIH 3T3 assay. These data demonstrate that the active cellular oncogene is not present in the hepatic tissue via a germ-line transmission but is activated only in those cells of the tumor tissue. Experiments using Southern blot hybridization analysis have identified this active cellular oncogene to be a member of the ras oncogene family. Identification of this cellular oncogene will now allow the evaluation of factors which might modify its expression. These future studies will lead to an increased understanding of potential mechanisms by which hepatic tumors are enhanced and should provide more informed estimates of risk for man based on bioassay data generated in this strain of mouse.

Chemical transformation of mouse liver cells results in altered cyclin D-CDK protein complexes.

Dysregulated cell proliferation is one phenotypic change associated with neoplasia. Key protein complexes involved in regulating cell division are composed of cyclins, cyclin-dependent kinases (CDK) and CDK inhibitors (CDI). Many virally transformed cells in culture exhibit disrupted cyclin-CDK-CDI complexes, suggesting that such changes may play a mechanistic role in viral transformation. To determine whether similar alterations may be involved in chemical carcinogenesis we characterized cyclin D1-CDK-CDI protein complexes in a non-tumorigenic mouse liver cell line and investigated whether complexes were altered after transformation with the genotoxic carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 3-methylcholanthrene (MC). In non-tumorigenic mouse liver cells cyclin D1 associated with CDK6, CDK4 or CDK2 to form binary (cyclin D1-CDK), tertiary (cyclin D1-CDK-p27KIP1) or quaternary (cyclin D1-CDK-p21WAF1-PCNA) complexes. After chemical transformation of mouse liver cells with either MC or MNNG, select cyclin D1-CDK-CDI protein complexes were altered. In MC-transformed cells formation of various binary, tertiary and quaternary cyclin D1-CDK-(CDI) protein complexes was reduced, resulting in decreased CDK4 kinase activity. Interestingly, CDK6 kinase activity was dramatically elevated due to high levels of cyclin D3 in association with CDK6. In MNNG-transformed cells select cyclin D1-CDK6-CDI and cyclin D1-CDK2-CDI protein complexes were altered but CDK6 and CDK4 kinase activity remained unaffected. Distinct changes in cyclin D1-CDK-CDI complexes found between the two chemically transformed mouse liver cell lines suggest that each cell line harbored unique mutations or alterations that differentially contributed to stabilization of cyclin D1-CDK-CDI holoenzymes. p53 gene mutations were not detected in the MC- or MNNG-transformed mouse liver cell lines and thus were not involved in disrupting cyclin D1-CDK-CDI protein complexes. In summary, this study presents evidence that D-type CDK protein complexes can be altered physically and functionally after chemical transformation with genotoxic carcinogens, suggesting that components of the cell cycle machinery can be targeted during chemical carcinogenesis.

Scanning transmission ion microscope with a field ion source.

Experiments with a low-resolution scanning transmission ion microscope, using hydrogen ions from a field ionization source, indicate that it will be feasible by this approach to aim at high-resolution ion microscopy. Micrographs of unstained biological specimens have been obtained by critical range absorption of a 55 keV hydrogen ion beam at a resolution of 2000 A.

Altered gene expression in spontaneous hepatocellular carcinomas from male B6C3F1 mice.

In this study, we analyzed spontaneous hepatocellular carcinomas (HCCs) from male B6C3F1 mice for alterations in the expression of the genes for c-myc, insulin-like growth factor II (IGF-II), cyclin D1, transforming growth factor-alpha (TGF-alpha), and the epidermal growth factor receptor (EGFR). These genes are all important in growth control in the rodent liver, and therefore, alterations in these genes or their products may result in unregulated growth. Northern blot analysis demonstrated an increase in expression of c-myc mRNA in five of 21 (24%) spontaneous HCCs compared with nontumor tissue. Tumors that had an increase in c-myc mRNA did not have an amplified c-myc gene. Of the HCCs analyzed, 18 of 29 (62%) showed reexpression of IGF-II RNA when compared with controls. Cyclin D1 mRNA was overexpressed in seven of 27 (26%) of the tumors analyzed relative to controls. Tumors with an increase in cyclin D1 mRNA also overexpressed the cyclin D1 protein. RNA encoding for the EGFR was decreased in 21 of 23 (91%) HCCs when compared with controls. None of the 29 liver tumors analyzed for alterations in expression of TGF-alpha mRNA differed from controls. Also, each individual tumor had a unique set of molecular alterations even when different tumors from the same animal were analyzed. These novel findings suggest that IGF-II, cyclin D1. c-myc, and EGFR are important mediators of carcinogenesis in spontaneous mouse liver tumor formation.

Genomic instability, as measured by microsatellite alterations, is not associated with liver tumor development in the genetically susceptible B6C3F1 mouse.

Certain human heritable forms of colon cancer have characteristically high frequencies of microsatellite alterations. These microsatellite changes are markers of genomic instability and the direct consequence of mutations in genes involved with DNA mismatch repair processes, which are in part responsible for maintaining the sequence integrity of the genome. Given that the B6C3F1 mouse is genetically predisposed to develop liver tumors we were interested in determining whether tumors derived in this strain of mouse may contain alterations in microsatellite sequences. The analysis of 48 tumors at 24 different microsatellite loci revealed that microsatellite alterations were detected in 12 of 48 tumors (25%). Although this frequency is relatively high, 11 of the 12 tumors exhibited only a single alteration and in 10 of those tumors this change was at the same microsatellite locus. Microsatellite alterations were also detected in the DNA isolated from 6 of 22 (27%) normal liver tissues with 4 of the 6 occurring at the same locus where the majority of changes were observed in the tumors. Based on these results, we conclude that the microsatellite alterations present in the mouse liver tumor tissue are most likely the result of spontaneous mutational events. Consequently, the genomic instability operational in a particular type of hereditary human colon cancer does not appear to be operational in the genetically predisposed B6C3F1 mouse liver. In addition, we demonstrated that the activation of the H-ras gene, which causes some forms of genetic instability in vitro, does not contribute to genetic instability within liver tumors as measured by microsatellite alterations.

Reverse transcription-polymerase chain reaction-based methodology to quantify differential gene expression directly from microdissected regions of frozen tissue sections.

Quantitative differences in the expression of oncogenes are a critical feature of the cancer process. Several methods are currently available for assessing differential gene expression, but none can be used to determine quantitative changes in gene expression from small numbers of cells. The ability to conduct this type of quantitative analysis would be useful in the study of definable, early stages of carcinogenesis when very few cells are involved. We therefore developed a highly sensitive, slide-based technique that incorporates the benefits of in situ polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) to quantify differential c-myc gene expression from liver tissue sections having either low or high levels of proliferating hepatocytes. To eliminate the need for isolating and quantifying mRNA, cells of interest were microdissected from frozen histological sections and their RNA directly subjected to RT-PCR amplification. These reactions were conducted in the presence of an internal RNA standard that was specifically designed to normalize differential RT and PCR efficiencies between samples. GENESCAN software analysis was used to determine the ratios of the RT-PCR products of the target gene to the RNA standard. These ratios were then normalized to the numbers of cells isolated, as quantified by image analysis, and comparative gene expression values were determined between sample groups. We conclude that this technology can be adapted to study any gene of interest in any type of frozen tissue or isolated cells. This methodology is particularly applicable to the molecular analysis of histopathologically distinct preneoplastic and neoplastic lesions identified on tissue sections.