Carcinogenicity of dietary dimethylnitrosomorpholine, N-methyl-N'-nitro-N-nitrosoguanidine, and dibromoethane in rainbow trout.

Eighteen-mo feeding trials of rainbow trout were used to test the carcinogenicity of 5 chemicals in this species. A single exposure level was used for each substance. The doses and chemicals tested were 1,556 ppm 2,6-dimethylnitrosomorpholine (DMNM), 500 ppm N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2,000 ppm 1,2-dibromoethane (DBE), 2,000 ppm 1,1-dichloroethylene (DCE), and 200 ppm cyclophosphamide (CP). Liver and/or glandular stomach neoplasms were produced by DMNM (liver and stomach), MNNG (stomach), and DBE (chiefly, stomach tumors). In addition, DMNM produced a low incidence of swimbladder papillomas and caused testicular atrophy in 50% of treated males. DCE and CP produced no neoplasms at the exposure levels used. No evidence of other chronic toxicity was seen for any of the 5 compounds.

Dose-dependent carcinogenicity and frequent Ki-ras proto-oncogene activation by dietary N-nitrosodiethylamine in rainbow trout.

While the experimental data upon which current concepts in mechanistically based risk assessment and molecular epidemiology are grounded derive almost entirely from rodent models, fish models have several attributes (e.g., low background incidence, extremely low cost tumor studies, nonmammalian comparative status for extrapolation of mechanisms to humans) that make them valuable adjuncts for addressing these concepts. This report provides an initial characterization of the dose dependency of dietary N-nitrosodiethylamine (DEN) hepatocarcinogenicity in Shasta strain rainbow trout (Oncorhynchus mykiss) and the potential of DEN to elicit ras proto-oncogene activation in this species. Carcinogen was administered in the diet at five concentrations for 12 months. Necropsies were performed at 9, 12, and 18 months, the latter on fish maintained on control diet for 6 months after cessation of DEN exposure. The incidence of hepatic neoplasms at the lower dietary concentrations (< or = 70 ppm) did not consistently exceed that for control groups, which were higher in this particular study (2%) than expected (historically 0.1%). For the higher DEN concentrations, a linear relationship between the hepatic tumor incidence (expressed as log odds, log [p/(1-p)], where p = proportion of fish bearing tumors), and the logarithm of total cumulative dose was observed, with response being independent of the length of time (9 or 12 months) during which the dose was accumulated. The dose-response curve for fish maintained an additional 6 months postexposure was shifted toward higher incidence but was parallel to the curve for fish killed at cessation of exposure. The model predicts that doubling the dose will produce somewhat more than a doubling of the odds (p/(100-p)) for tumor incidence and that the odds for lesions 6 months postexposure will be approximately double those at cessation of exposure. Comparison of these results with previous studies using rats suggests an overall similarity in dose-response curves, with trout being somewhat less sensitive than rats to DEN hepatocarcinogenesis. To examine the molecular basis for DEN carcinogenesis in this species, seven liver tumors induced separately by short-term DEN treatment were probed by 3'-mismatch primer polymerase chain reaction analysis for evidence of Ki-ras proto-oncogene activating point mutations. A very high proportion (6/7) of tumors was found to carry codon 12 GGA-->AGA mutations, whereas no codon 61 mutants were detected in this sample.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative effect of dietary butylated hydroxyanisole and beta-naphthoflavone on aflatoxin B1 metabolism, DNA adduct formation, and carcinogenesis in rainbow trout.

Butylated hydroxyanisole (BHA) and beta-naphthoflavone (BNF), both chemicals with anti-carcinogenic properties in some experimental animals, were compared for effects on aflatoxin B1 (AFB1) metabolism, hepatic DNA adduct formation and carcinogenesis in the rainbow trout. Dietary BHA had no effect on the hepatic tumor incidence when fed at 0.03 or 0.3% 4 weeks prior to and during a 4 week dietary exposure of 10 p.p.b. AFB1. BNF, when fed at 0.005 or 0.05% under similar conditions, significantly reduced tumor response, which confirms previous results in trout (Nixon et al., Carcinogenesis, 5, 615-619, 1984). BHA fed at either 0.03 or 0.3% for 8 weeks had no post-initiation effect on the 52 week hepatic tumor incidence of trout exposed to a 0.5 p.p.m. AFB1 solution as embryos. A similar post-initiation exposure to 0.05% BNF significantly enhanced AFB1 tumor response. The influence of dietary BHA and BNF on AFB1 metabolism and DNA adduct formation and persistence in trout were examined. A 3 week pre-treatment with 0.3% dietary BHA had no effect on in vivo hepatic nuclear AFB1-DNA adduct formation at 0.5, 1, 2 and 7 days after AFB1 i.p. injection. By contrast 0.05% dietary BNF reduced hepatic AFB1-DNA adducts to 33-60% of control levels at 0.5, 1, 2 and 4 days after AFB1 exposure. This was accompanied by significantly lower blood and liver levels of AFB1 during the first 24 h after i.p. injection. Livers of BNF trout also contained 4-fold more of the less carcinogenic metabolite, aflatoxin M1, and 50% less aflatoxicol (AFL), a metabolite with similar carcinogenicity as AFB1. Bile AFL-glucuronide levels were significantly decreased in BNF-fed trout, but total bile glucuronides were significantly increased due to a 15-fold increase in aflatoxicol-M1 glucuronide. Freshly isolated hepatocytes from BHA-fed fish, when incubated with AFB1 for 1 h, showed no difference in levels of AFB1-DNA adducts or ratios of AFB1 metabolites when compared to hepatocytes isolated from fish fed a control diet only. By contrast, dietary BNF has been previously shown to greatly enhance AFM1 production, reduce AFL production, and significantly reduce AFB1-DNA adduct formation in isolated trout hepatocytes (Bailey et al., Natl. Cancer Inst. Monograph, 65, 379-385, 1984). These results indicate that dietary BHA up to 0.3% does not alter AFB1 metabolism or DNA adduction in trout, nor does it inhibit or promote AFB1 hepatocarcinogenesis in this species.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of carcinogenesis by the natural anticarcinogen indole-3-carbinol.

Indole-3-carbinol (I3C), a natural constituent of cruciferous vegetables, is an inhibitor in several experimental animal models of carcinogenesis by polynuclear aromatic hydrocarbons or aflatoxin B1 (AFB1) when administered prior to or during carcinogen exposure. For assessment of the postinitiation effects of I3C, rainbow trout were exposed to dietary I3C in two different protocols--before and during AFB1 exposure or after AFB1 exposure only. Preinitiation exposure to I3C reduced AFB1-initiated hepatocellular carcinomas in trout as previously reported, but post-initiation I3C exposure strongly enhanced the tumor incidence above the positive AFB1 control. These results reveal the need for additional research to elucidate the overall effect of I3C on chemical carcinogenesis.

Mechanisms of anti-carcinogenesis by indole-3-carbinol: effect on the distribution and metabolism of aflatoxin B1 in rainbow trout.

Indole-3-carbinol (I3C), a component of cruciferous vegetables, was previously shown to inhibit aflatoxin B1 (AFB1) carcinogenesis in trout. The purpose of this study was to examine the effect of I3C on AFB1 metabolism and hepatic DNA adduct formation in vivo and in vitro. When fed at 0.2%, I3C produced a 70% reduction in average in vivo hepatic DNA binding of injected AFB1 over a 21-day period when compared to controls. A 24-h distribution study of injected tritiated AFB1 in I3C fish showed less total radioactivity in the blood and liver at all times examined, compared to controls. These reductions were due primarily to reduced levels of AFB1 bound to red blood cell DNA, reduced plasma levels of the primary metabolite aflatoxicol (AFL), and decreased levels of AFB1 and polar metabolites present in the liver of I3C fish. In contrast to blood, total radioactivity was significantly elevated in the bile of I3C fish resulting from a 7-fold increase in aflatoxicol-M1 glucuronide levels over controls. No difference was observed in concentration of AFL glucuronide, the primary conjugate present in control fish. There was no difference in total radioactivity remaining in the carcass of I3C or control fish. AFB1 metabolism in freshly isolated hepatocytes from I3C fish showed 20% less DNA binding in a 1-h assay, with a 2-fold increase in aflatoxin M1 production. Addition of I3C to control hepatocytes at levels of 1, 10 or 100 microM had no effect on AFB1 DNA binding. These findings indicate that I3C inhibition of AFB1 hepatocarcinogenesis in trout involves substantial changes in the pharmacokinetics of carcinogen distribution, metabolism and elimination, leading to significantly reduced initial hepatic-nuclear DNA damage in vivo.

Complications following removal of impacted third molars: the role of the experience of the surgeon.

The incidence of complications associated with the removal of impacted third molars in a group of 500 patients treated by oral surgery faculty were compared with the incidence of complications in 208 patients treated during the same period by residents of oral and maxillofacial surgery. The results show that complications were more numerous after the removal of third molars classified as partial bony or complete bony impactions, and that less-experienced surgeons had a significantly higher incidence of such complications.

Mechanisms of anti-carcinogenesis: the distribution and metabolism of aflatoxin B1 in rainbow trout fed aroclor 1254.

Polychlorinated biphenyls (PCBs) have previously been shown to be inhibitors of carcinogenesis in trout. The mechanism of this inhibition was investigated by studying the effects of PCBs on aflatoxin B1 (AFB1) distribution, metabolism and DNA adduct formation, both in vivo and in vitro. A 24 h distribution study of injected tritiated AFB1 showed more radioactivity in blood, liver and bile in fish fed PCBs, but less in residual carcass. The metabolites of AFB1 found in vivo in blood plasma and liver homogenates were shifted by PCB pretreatment towards greater production of the polar metabolite aflatoxin M1 (AFM1) and glucuronide conjugates. The major metabolite in bile of PCB fish was the glucuronide of aflatoxicol M1 (AFL-M1), which was enhanced 15-fold over controls. Levels of aflatoxicol (AFL) glucuronide, the major conjugate in controls, were unaltered by PCBs. The pattern of AFB1 metabolism in isolated hepatocytes from PCB-prefed fish was consistent with in vivo metabolism. AFB1--DNA adduct formation in a 1 h assay was similar in hepatocytes from PCB-fed and control fish. However, the total rate of AFB1 metabolism was significantly elevated in hepatocytes from PCB-fed fish such that the degree of AFB1--DNA adduct formed per unit AFB1 metabolized was 42% lower than control. Similarly, adduct formation in vivo during the first 24 h post-AFB1 injection in PCB fish was not significantly different from controls. However, over a longer 21 day period, adduct levels in PCB fish were only 48-69% of controls (P less than 0.005, analysis of variance), once peak adduct formation was reached. Thus, initial rates of adduct formation may be misleading in the absence of further information on rates of carcinogen metabolism in vitro and/or pharmacokinetics of peak adduct formation in vivo. These results indicate that PCB inhibition of AFB1 carcinogenesis in trout involves dramatic initial changes in carcinogen distribution, metabolism and elimination which, over time, results in a net reduction of DNA adduct formation.

Hepatocarcinogenicity of benzo[a]pyrene to rainbow trout by dietary exposure and intraperitoneal injection.

The influence of benzo[a]pyrene [(BP) CAS: 50-32-8] on the induction of certain enzymes within the hepatic mixed-function oxidase (MFO) system and its potential carcinogenicity were examined in rainbow trout (Salmo gairdneri). Nine-week feeding trials were performed with 500 and 1,000 ppm BP to determine trout tolerance to BP. Levels of MFO enzymes, including ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD), benzo[a]pyrene monooxygenase (BPMO), and cytochrome P450 were measured during this time. An 18-month feeding trial of a 1,000-ppm BP dose was initiated in duplicate groups of 100 fingerling rainbow trout. Samples of trout were killed at 6, 12, and 18 months for gross and histologic examination of the internal organs for neoplasms. A group of fifty 10-month-old rainbow trout were given 12 monthly ip injections of 1 mg BP in 0.4 ml propylene glycol (PG), and comparable controls were given PG injections only. The trout were held for an additional 6 months, killed at age 28 months, and examined as in the dietary study. Mean MFO enzyme levels of EROD, ECOD, BPMO, and cytochrome P450 were significantly (P less than .001) elevated, showing dose- and time-response relationships when compared to MFO enzyme levels in control fish. Twelve months after BP exposure was initiated, 15% of the BP-fed fish had histologically confirmed neoplasms of the liver. After 18 months the incidence increased to 25%. No evidence of neoplasia was observed in control fish. BP injected ip resulted in a 50% incidence of hepatocellular neoplasms and in a fibrosarcoma of the liver and papillary adenomas of the swim bladder in 1 fish. These results indicate that BP is a potent inducer of selected hepatic MFO enzymes and establish, for the first time, the hepatocarcinogenicity of BP in an aquatic species.

The hepatocarcinogenicity of diethylnitrosamine to rainbow trout and its enhancement by Aroclors 1242 and 1254.

Rainbow trout (Salmo gairdneri) were fed diets containing 100 ppm Aroclor 1242 (AC42) or Aroclor 1254 (AC54) in combination with 1100 ppm diethylnitrosamine (DEN) for one year. The incidence of hepatocarcinomas was determined and compared with the incidence in trout fed 1100 ppm DEN alone. The two Aroclors dramatically enhanced tumor incidence from 10.2% in the positive controls (DEN alone) to 40.2% for AC42 and 21.6% for AC54. This is in contrast to previous results obtained when AC54 was fed concomitantly with aflatoxin B1 (AFB1), where a substantial inhibition of carcinogenesis was observed. The alteration of chemical carcinogenesis in trout by PCB, therefore, depends upon the carcinogen involved and is not a generalized effect.

Histological progression of hepatic neoplasia in rainbow trout (Salmo gairdneri).

The histological progression of hepatic neoplasia has not been as systematically studied in rainbow trout as it has been in rodents. Two putative preneoplastic lesions have been identified, the eosinophilic focus and the basophilic focus, but whether these correspond to similar lesions in rodent livers is not known. Preneoplastic liver lesions in rodents have been extensively characterized histochemically, but adaptation of these techniques to trout livers has not always been successful. Eosinophilic foci consist of hypertrophied cells, enlarged atypical nuclei, and dense glycogen-free cytoplasm. Mitotic figures are also occasionally seen. Usually, these foci have been infiltrated and at least partially destroyed by inflammatory cells, largely lymphocytes. In some liver sections, eosinophilic foci are intact and occasionally an eosinophilic-basophilic transformation can be seen. However, most often basophilic foci appear independently, surrounded by normal hepatocytes, with no indication of a prior eosinophilic stage. The cells of basophilic foci are similar to those of carcinomas: intensely basophilic, mitotically active, devoid of glycogen, and grouped into cords several cells in thickness. These nodules may appropriately be referred to as carcinomas in situ, because the only distinguishing characteristic is the size of the lesion. Attempts at differentiation between benign and malignant liver lesions appear arbitrary. We believe the best classification of the neoplastic liver lesion in trout is a hepatocellular carcinoma because the potential for malignant behavior always exists and, with sufficient time, can often be histologically demonstrated. We have also described our experience with the characteristics of other liver lesions associated with hepatocarcinogenesis.

Effect of dose on the inhibition of carcinogenesis/mutagenesis by Aroclor 1254 in rainbow trout fed aflatoxin B1.

Prior studies have shown that Aroclor 1254 (PCB) differentially alters the incidence of aflatoxin B1 (AFB1) induced hepatocellular carcinomas in trout, depending upon the time of PCB administration relative to AFB1 exposure (Shelton et al., 1983). When fed simultaneously with AFB1, PCB inhibits carcinoma incidence. We investigated the effect of AFB1 and PCB dose on this inhibition. Duplicate tanks of 100 rainbow trout were fed AFB1 at concentrations of 1, 4, or 8 ppb, either with or without the addition of 50 ppm PCB. Other groups were fed 4 ppb AFB1 + 5 ppm PCB, 50 ppm PCB alone, or control diet alone. After 9 and 12 mo, 40 and 60 fish per tank, respectively, were sampled to determine the incidence of liver tumors. The results show a parallel inhibition of the AFB1-tumor dose-response curve by the presence of 50 ppm PCB. Fish fed 4 ppb AFB1 + 5 ppm PCB showed slight inhibition in response when compared with 4ppb AFB1 alone. Also, livers from fish fed 50 ppm PCB were used to prepare S20 for use in the Salmonella mutagenesis assay. These livers were less efficient in converting AFB1 to a mutagen, when compared to control S20. The AFB1-mutagenesis dose-response curve was again shifted parallel to the right of the curve generated using control S20. These results suggest that the inhibitory action is at least partly at the level of carcinogen activation. The finding of parallel, as opposed to proportional, inhibition with varying carcinogen exposure for certain classes of inhibitors may have important implications for inhibition of environmental carcinogenesis at low levels of carcinogen exposure.

Effects of dietary aroclor 1254 and cyclopropene fatty acids on hepatic enzymes in rainbow trout.

Diets containing Aroclor 1254 (PCB); cyclopropene fatty acids (CPFA), and PCB plus CPFA were fed to rainbow trout (Salmo gairdneri) for 15 weeks to determine the effects on hepatic microsomal activities of ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, and benzo(a)pyrene monooxygenase. Ethoxyresorufin-O-deethylase activity continued to increase to a level 77-fold higher than control at week 15. Ethoxycoumarin O-deethylase and benzo(a)pyrene monooxygenase activities increased to 7.1-fold and 47-fold over control at week 9, respectively. Cytochrome P-450 values remained approximately 2-fold above controls from week 5 through week 15. At weeks 1 and 3, cytochrome P-450 levels were not significantly different from control. Dietary CPFA significantly depressed ethoresorufin-O-deethylase and ethoxycoumarin-O-deethylase activities, but had no effect on benzo(a)pyrene monooxygenase activity. Ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, nd benzo(a)pyrene monooxygenase activities in the combined PCB-CPFA-fed trout were significantly higher than in control- or CPFA-fed trout, and significantly lower than in PCB-fed trout. This is the first time dietary PCBs have been shown to induce the MFO system in rainbow trout. These results provide a possible explanation for the effects of dietary PCBs on the metabolism and expression of other chemical carcinogens.

Comparative mutagenicity of aflatoxins using a Salmonella/trout hepatic enzyme activation system.

A modification of the Ames assay using rainbow trout (Salmo gairdneri) liver postmitochondrial fraction (PMF) was developed to investigate the relative mutagenic potential of a series of aflatoxins (AFs). Preliminary experiments revealed that the 20000 x g (S20) liver fraction contained a higher metabolic activity than either the S9 or S30 fractions, and that 5 mg of S20 protein/plate gave the highest mutagenic response. A 9-24 h preincubation period at 25 degrees C was also required. The results from comparative mutagenicity experiments showed the following relative potencies: AFB1 greater than AFL greater than AFG1 greater than AFM1 greater than AFB2 greater than AFP1 greater than AFQ1. The relative potencies observed with this in vitro system qualitatively correlated with the in vivo carcinogenic activity seen in trout, indicating that this assay is of value in predicting the carcinogenic potential of mycotoxins in this species.

Treatment of maxillary arch collapse in an adult cleft palate patient: report of case.

The combined orthodontic, orthognathic surgical, and prosthodontic reconstruction of the previously treated bilateral cleft lip and cleft palate of a 20-year-old white man is reviewed. Maxillary arch collapse and severe malocclusion occurred after palatal closure in early childhood with alveolar bone grafting and premaxillectomy. A modified Le Fort I osteotomy, using a jackscrew with a modified posterior hinge, was employed to achieve rapid palatal expansion. In addition, mandibular subapical osteotomy was performed, in conjunction with postoperative orthodontic realignment. Fixed prosthodontic restorations were then used to achieve stability and a more esthetic appearance.

Expansion meshed skin grafts in care of the donor site in skin grafting vestibuloplasty.

A method of caring for split-thickness skin graft donor sites using a physiologic dressing of meshed donor material, taken at the same time and location as the split thickness skin graft harvested for maxillary or mandibular vestibuloplasty, has been presented. This procedure is simple to accomplish and provides rewarding postoperative results, with rapid re-epithelialization of the donor site, early ambulation, absence of pain, and an excellent final cosmetic result.

Spontaneous-ventilation general anesthesia for outpatients using enflurane.

We have described our technique for general anesthesia for outpatients using a mixture of nitrous oxide, oxygen, and enflurane with spontaneous ventilation through a Bain anesthetic circuit. In our experience, surgical excision of impacted teeth, multiple extractions and alveoloplasty, enucleation of simple cysts, excision of benign tumors, and other similar operations have been done efficiently with these anesthetic techniques. More than 500 such cases have been managed without significant complication. The concepts of selection and preparation of patients, airway security and protection during surgery performed in the upper airway on an unconscious patient who is breathing spontaneously, continuous monitoring of vital functions, and rapid induction and recovery all combine to make this technique consistent with standards for excellence in anesthesia.