bluuebunny/crossref_metadata_2025 huggingface dataset:
Design of Carcinogenicity Studies: A Backward Glance
Authors: Diener Robert
Published: September 2009

Despite extensive research carried out over many years, a completely satisfactory method of testing drugs and chemicals for carcinogenicity continues to elude toxicologists. In the past, numerous scientific groups, regulatory agencies, and regional bodies have discussed and published specific recommendations and guidelines regarding the testing requirements for carcinogenicity studies. The Food, Drug and Cosmetic Act became law in 1938, but it was not until after the Second World War that serious concerns regarding chemicals became prevalent. Testing guidelines were nonexistent until 1949 when the FDA published “Procedures for the Appraisal of the Toxicity of Chemicals in Foods.” This key publication was the forerunner of the famous “Gray Book” (1959) in which were outlined many of the specific carcinogenicity testing procedures still used today. Guidelines and regulations became the watchwords during the 60's and 1970's. Formation of the EPA, more stringent testing requirements, Toxic Substances legislation, and Environmental Control Acts followed one another in short order. NCI's Technical Report #1 (1976) entitled, “Guidelines for Carcinogen Bioassay in Small Rodents” probably had the greatest impact of any publication on carcinogenicity testing. Many later guidelines and recommendations were based on this publication.
Design of Carcinogenicity Studies, Dose Selection, Route, Blood Levels, Transformation
Authors: Neal Robert A.
Published: September 2009

The results of subacute toxicity tests are the information most commonly used for setting doses for carcinogenicity studies. However, since diet composition is important in regard to tumor incidence, knowledge of the effect of various doses of the compound on the nutritional composition and quality of the diet of the animals on test is also important. It is also important to determine if any of the doses administered in the study exceed the ability of the species under study to excrete it in a manner which is approximately the same as that seen at lower doses. Similarly, it is important to determine if the higher doses administered lead to the formation of metabolites not seen at lower doses which are more nearly equivalent to those to which humans are or will be exposed. The compound under test should, if possible, be administered by the same route by which human exposure occurs or is anticipated to occur. Because of potential difficulties in the interpretation of the results, gastric intubation is, in general, the least desirable method of administration. In selecting the most appropriate animal species to be used in a carcinogenicity study, consideration should be given to the use of the species and strain of experimental animal that is most biologically and metabolically similar to humans, if such data are available.
Dose Selection as it Pertains to Testing a Prodrug in Carcinogenicity Bioassays
Authors: Szczech George M. , Tucker Walter E.
Published: September 2009

Toxicologists need more information than is usually available in the early stages of development of a drug in order to choose proper dose levels for testing in the bioassays. The approach most likely to result in successful bioassays involves an early multidisciplinary effort in which there is pharmacokinetic characterization of the test material in both rats and mice. Preliminary 3 month studies are desirable. Periodic sampling of plasma is essential to detect possible non-linear kinetics (as in the example we report herein) reflected as accumulation of the test material or metabolites. This is true regardless of the test substance. However, if one tests prodrugs it may be particularly helpful to know if chemical or enzymatic conversion of the prodrug is linear and if there is reversion to prodrug or other abberant metabolism. Failure to rule out these possibilities could result in subsequent clinically irrelevant organ damage or could compromise longevity or the interpretation of results in lifetime studies. Pharmacokinetic considerations are as valid as the more traditional biologic or morphologic end points used to estimate maximum tolerated or no-effect dose levels.
Pathology Requirements for Rodent Two-Year Studies I. A Review of Current Procedures
Authors: McConnell Ernest E.
Published: September 2009

In the early 1970s the National Cancer Institute (NCI) initiated an animal bioassay program to determine if chemicals, to which the public are exposed, are hazardous by nature of their ability to induce cancer in animals. Experience with such tests led to the development of program guidelines in 1976. The pathology portion of this protocol included the histopathologic examination of a minimum of 25 organs/tissues from every animal on study. Since 1976 additional tissues have been added so that the current National Toxicology Program (NTP) protocol includes the histopathologic examination of 42-44 sections of 31-33 tissues from each animal. Thus an average of 25,000-26,000 sections are evaluated on a typical 600 animal (300 rats and 300 mice) study. A review of 277 NCI/NTP chemical bioasssys which used this protocol revealed that this extensive list appears to be in excess of what is required to answer the question of whether a given chemical causes cancer in the test species. In addition, this protocol may be flawed in its ability to detect non-neoplastic chemically related disease.
Statistical Issues in the Design and Analysis of Carcinogenicity Bioassays
Authors: Hajian Gerald
Published: September 2009

A general outline of the statistical issues in the design and analysis of carcinogenicity bioassays is given in this paper. Design issues, such as assignment of animals to treatment groups, dual control groups, duration of study, and the number of animals per group are discussed. Information needed by the biostatistician are listed to facilitate the recording of data by the pathologist. Issues in the analysis of tumor incidence data are given. Use of historical control data is encouraged and discussed.
Spontaneous Hypertrophic Foci of Pancreatic Acinar Cells in CD Rats
Authors: Chiu Taisan
Published: September 2009

A study was conducted to determine the spontaneous occurrence of hypertrophic foci of the pancreatic acinar cells in rats. Tissues from untreated control rats, 4 to 27 months of age and used in either subchronic and chronic feeding toxicity or carcinogenicity studies were reviewed. The lesions, morphologically distinctly different from nodular hyperplasia or hyperplastic nodules and adenomas, were microscopic in size and occurred in 109 of 607 males (17.6%) and 87 of 517 females (16.8%). The overall incidence of hypertrophic foci in the pancreas from rats of both sexes significantly increased with age. The lesion was not sex-related. Neither the size nor the number of lesions appeared to increase with age. The lesion represents a distinct pathologic entity and is considered to be neither hyperplastic nor neoplastic.
Pathologist's Responsibility in the Diagnosis of Oncogenesis
Authors: Iatropoulos Michael J.
Published: September 2009

In recent years with all sophisticated incoming data, it has become obvious that “cancer” is indeed a cellular disease. This level of biological organization is extremely pivotal, because it is at this level that the interactive control mechanisms have failed, rendering excessive proliferation and uncoordinated growth, which persists after cessation of the stimuli which evoked it. Although we are extremely interested in identifying and describing the stimuli (etiology), presently as toxicologic pathologists, we are called upon in rendering a diagnosis as to whether given findings are “cancer”, and by extension whether an administered substance has caused these findings in laboratory animal species. For some time now the etiology has been confused with the diagnosis and the identification of the causative stimuli with the manifested effects. Although we don't know yet the entire story about the etiology of the oncogenic process we can say that we know a lot about its manifestations and therefore we are capable of making the diagnosis. By virtue of our training, we are capable of identifying, classifying and describing all oncogenic manifestations, no matter whether they are in mice or men. In the course of this pursuit we need some order. Oncogenesis is a multistep, multistage process. We must therefore have a design capable of describing and differentiating initiation, potentiation, promotion, modification, and autonomy in our laboratory animal studies. We must also address the status of the biological resistance of the animal organism through pertinent comparative correlations. Furthermore, since “cancer” is a disease, and a syndrome, we must not limit ourselves in studying only the incidence of a single manifestation in an animal, especially if we intend to use it in extrapolation, but the entire multiplicity of manifestations. In biology, in order to effectively extrapolate, we need appropriate changes in the orders of magnitude. The new chemical entity should be compared structurally to the known human and rodent oncogenic substances. The interspecies differences in toxicokinetics and metabolism compared and the type and location of target cell, as well as, the extent of genetic distortions should be established. By implementing all these it is possible not only to render an accurate diagnosis but also estimate the human “cancer” risk.
Chemical Carcinogens Without Mutagenic Activity: Peroxisome Proliferators As A Prototype
Authors: Reddy Janardan K. , Scarpelli Dante G. , Subbarao Vadrevu , Lalwani Narendra D.
Published: September 2009

Chemical carcinogens can be classified into two categories (i.e. mutagenic and non-mutagenic) on the basis of positive or negative evidence of DNA damage, mutagenicity or chromosomal aberrations in short-term test systems. Evidence indicates that carcinogenic peroxisome proliferators are negative in short-term test systems. This paper outlines approaches which may be useful in identifying a chemical carcinogen without mutagenic activity. It is conceivable that an alteration in DNA, if essential for initiation of neoplasia, may be mediated indirectly by the biological effects of nonmutagenic carcinogens.
Non-Reversibility of Vinyl Chloride Carcinogenesis in Rodents
Authors: Bhandari Jagdish C.
Published: September 2009

A review of the data obtained from various studies on carcinogenicity of vinyl chloride (VC) in rodents, particularly on the effect of dose, age, duration of exposure and potential reversibility of lesions, revealed that vinyl chlorideinduced carcinogenicity in rodents was dose and time related; no recovery occurred in mice even after only 1 month of VC exposures or in rats after 6-month exposures. In addition, younger animals (2 months old) were more susceptible to VC-induced carcinogenicity than animals held for 6 or 12 months prior to exposure. Initial 6 or 12 month exposures were adequate to detect the carcinogenic potential of VC. The above information was used as a basis for discussion on design of carcinogenicity studies. Possibility of determining the carcinogenic potential of a compound in a shorter period than the traditional 2 year studies in rodents was discussed in consideration with appropriate doses, species, age and exposure duration. Although this approach may be applicable to a strong carcinogen, it was not considered practicable in case of weak or unknown carcinogens.
Morphological Effects of Subchronic Oral Sulfamethazine Administration on Fischer 344 Rats and B6C3F1 Mice
Authors: Heath James E. , Littlefield Neil A.
Published: September 2009

One hundred and forty-four Fischer 344 rats and 144 B6C3F1 mice (72/sex/species) were fed either a control diet or a diet containing 300, 600, 1200, 2400 or 3600 ppm sulfamethazine for 90 days. They were then necropsied and tissue specimens were evaluated for pathological changes by light and transmission electron microscopy. No gross or light microscopic lesions related to sulfamethazine administration were evident in the mice. Thyroid gland enlargement was evident at necropsy in one half of the rats (12 of 24) which received the 3600 ppm level of sulfamethazine and in 1 of 24 rats fed the 2400 ppm level. By light microscopy, thyroid gland hyperplasia was evident in rats which received all 5 dose levels of the compound, but this change was more pronounced and more frequent in those animals administered the higher concentrations. This thyroid hyperplasia was observed in rats of both sexes, but with greater incidence in males than in females, among the groups receiving the lower concentrations of compound. Ultrastructural changes included markedly dilated rough endoplasmic reticulum, altered microvilli, and diminished colloid droplets involving the thyroid follicular cells and compartmentalization of colloid within the follicular lumina.
Polychlorinated biphenyl (PCB) Toxicity in Adult Cynomolgus Monkeys (M. fascicularis): A Pilot Study
Authors: Tryphonas Leander , Truelove John , Zawidzka Zofia , Wong James , Mes Jos , Charbonneau Sylvia , Grant Donald L. , Campbell James S.
Published: September 2009

Aroclor 1254 and Aroclor 1248, at doses of 11.7 and 4.7 mg/kg body weight (equivalent to 5 and 2 mg/kg/day), were given 3 days per week to groups of cynomolgus monkeys, and caused weight loss, fingernail loss, facial edema, epiphora, and death. Blood and adipose tissue PCB concentrations rose with the length of treatment. Tissue concentrations in blood, adipose tissue, liver and kidneys were highest in monkeys treated with Aroclor 1254, reflecting dose differences. There was considerable variation, both within and between groups, in hematologic responses to PCB treatment. Aroclor 1254-treated monkeys had depressed and weakly responsive erythropoiesis. Aroclor 1248-treated monkeys had active but ineffective or depressed erythropoiesis with severe macrocytic or moderate normocytic anemia. Biochemical determination of blood serum constituents revealed treatment and time-related trends towards hypoalbuminemia and increased alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, lactic dehydrogenase, cholesterol, triglycerides, total bilirubin and direct bilirubin values. Pathologic lesions common in both Aroclor groups were dilatation of meibomian glands duct; mucinous hyperplasia of the gastric mucosa; atrophy and loss of germinal centers in the splenic and other lymphoid follicles; enlargement, fatty degeneration, and necrosis of hepatocytes; bile duct and gall bladder epithelial cell hypertrophy and hyperplasia; and thyroid aberrations in follicular cell size and number of intracytoplasmic lysosomes. Lesions seen exclusively in an Aroclor 1254-treated monkey were widespread mucinous metaplasia and hyperplasia of the fundic mucosa. The results suggest that in general, cynomolgus monkeys may be more refractory or less susceptible to PCB toxicity than rhesus monkeys and, that Aroclor 1248 may be more toxic than Aroclor 1254.
Subcellular Distribution of Styrene Oxide in Rat Liver
Authors: Pacifici Gian Maria , Cuoci Lulgi , Rane Anders
Published: September 2009

The subcellular distribution of (3H)-styrene-7,8-oxide was studied in the rat liver. The compound was added to liver homogenate to give a final concentration of 2 times 10−5; 2 times 10−4and 2 times 10−3M. Subcellular fractions were obtained by differential centrifugation. Most of styrene oxide (59-88%) was associated with the cytosolic fraction. Less than 15 percent of the compound was retrieved in each of the nuclear, mitochondrial and microsomal fractions. A considerable percentage of radioactivity was found unextractable with the organic solvents, suggesting that styrene oxide reacted with the endogenous compounds. The intracellular distribution of this epoxide was also studied in the perfused rat liver. Comparable results with those previously described were obtained. The binding of styrene oxide to the cytosolic protein was investigated by equilibrium dialysis and ultrafiltration. Only a small percentage of the compound was bound to protein.
Thioacetamide Effects on Protein Metabolism in the Liver: Lessons from Isolated Hepatocytes
Authors: Cajone Francesco , Bernelli-Zazzera Aldo
Published: September 2009

The effects of a short-term treatment with thioacetamide have been studied in isolated hepatocytes obtained from intoxicated rats. A technique has been developed which utilizes leucine alternatively labeled with either [14C] or [3H] and permits the simultaneous evaluation of protein synthesis, catabolism and secretion in the same cell during the same incubation period. The results indicate that short-term thioacetamide treatment causes an overall slowing-down of protein metabolism. Protein synthesis, however, decreases less than protein degradation and total protein secretion; albumin secretion, which is also less than normal, seems to be less compromised than total protein secretion.
Abnormalities in Liver Iron Accumulation During N-2-Fluorenylacetamide Hepatocarcinogenesis that are Dependent or Independent of Continued Carcinogen Action
Authors: Furuya Keizo , Maeura Yoshiichi , Williams Gary M.
Published: September 2009

The characteristics of liver iron accumulation were studied during N-2-fluorenylacetamide (FAA)-induced hepatocarcinogenesis in rats. After injection of iron-dextran in control rats, hepatocytes accumulated stainable iron evenly throughout hepatic lobules. During the feeding of FAA, iron accumulation was reduced in the midzonal and centrilobular regions. After FAA removal, hepatocytes in these regions again accumulated high amounts of iron. Hepatocellular altered foci induced by FAA displayed rather uniform (> 94%) iron-exclusion during FAA feeding. After FAA removal, however, iron-exclusion was lost in a fraction of the foci, while others (40-64%) remained resistant to iron accumulation. A large majority of liver neoplasms (> 93%) displayed resistance to cellular iron accumulation both during FAA feeding and after removal of FAA. Thus, iron-exclusion by liver neoplasms is carcinogen-independent and irreversible, in contrast with that of normal hepatocytes which is completely carcinogen-dependent and reversible. Altered foci appear to represent two populations: one is characterized by reversible iron-exclusion whereas the other, like neoplasms, possesses permanent iron-exclusion.
Nuclear Structure and Higher Order Gene Structure: Their Role in the Control of Chemically-Induced Neoplastic Transformation
Authors: Nicolini Claudio
Published: September 2009

An overview is hereby given of the physical and physicochemical alterations at the level of nuclear structures in rat liver cells, following a chemically-induced neoplastic transformation. These alterations refer to chromatin-DNA structure, from secondary (B- versus A- and Z-form) to tertiary-quanternary up to quinternary (in situ), and to the physical state of water. Possible molecular mechanisms-linking global chromatin changes to single gene expression in the control of neoplastic transformation-are discussed in terms of the degree of negative superhelicity of fibrosomes, recently identified single repeating structural sub-units and hereby associated with single functional genes.
Changes in Serum and Hepatic Cholesterol in Lead-Induced Liver Hyperplasia
Authors: Pani Paolo , Dessi Sandra , Rao Kalipatnapu N. , Batetta Barbara , Laconi Ezio
Published: September 2009

Lead nitrate when injected intravenously as a single dose to male Wistar rats causes a strong hepatic proliferative response followed by reabsorption of excess tissue within 10-14 days. The rate of cell proliferation in this hyperplastic model was positively correlated with hepatic de novo synthesis of cholesterol, stimulation of the hexose monophosphate shunt pathway of glucose metabolism and with alterations in serum lipoproteins.
Modulation of Radiation Induced Transformation by Combinations of a Phorbol Diester and a Lymphotoxin
Authors: DiPaolo Joseph A. , Doniger Jay , Popescu Nicholas C. , Evans Charles H.
Published: September 2009

The susceptibility of normal Syrian hamster embryo cells to transformation by environmental carcinogens has made possible the determination of a variety of responses as cells proceed to the neoplastic state. Expression of the initiated and promoted stages of irradiation carcinogenesis, for example, can be modified by cell surface alterations. Phytohemagglutin (PHA) or its isolectins decrease 12-O-tetradecanolphorbal-13-acetate (TPA) promoted transformation whereas PHA does not affect carcinogen only induced transformation. In contrast, both initiated and promoted transformation are sensitive to hamster lymphotoxin, a hormone-like, non-antibody lymphocyte glycoprotein. A 48 hr lymphotoxin treatment before or immediately after X-irradiation, or during TPA exposure causes a persistent inhibition independent of when carcinogen was added. A 6 hr lymphotoxin pulse before irradiation and TPA causes a persistent but non-permanent effect unless followed by the carcinogen treatment; lymphotoxin becomes more potent as the interval between the lymphotoxin pulse and carcinogen insult or TPA addition is reduced. PHA and lymphotoxin affect the biological activity of TPA by diverse mechanisms. PHA may alter either the binding of TPA to a critical cellular receptor for promotion or alter a later step in promotion. Lymphotoxin can prevent the initiation of transformation and modulate carcinogenesis as well at both initiated and promoted stages in the transition to the neoplastic state.
Orotic Acid, a New Promoter for Experimental Liver Carcinogenesis
Authors: Rao Prema M. , Nagamine Yoshiaki , Roomi M. Waheed , Rajalakshmi Srinivasan , Sarma Dittakavi S. R.
Published: September 2009

Male Fischer 344 rats initiated with 1,2-dimethylhydrazine 2HCl (100 mg/kg) given 18 hr after partial hepatectomy and exposed to a diet containing 1% orotic acid for 13 months developed a 100% incidence of hepatocellular carcinoma. The creation of nucleotide pool imbalances by dietary orotic acid, for e.g., an increase in uridine nucleotides and a decrease in adenine nucleotides, was considered as a possible mechanism for the promotional effect of orotic acid on liver carcinogenesis. The significance of this hypothesis is that altered nucleotide pools affect both genomic as well as membrane organization. Consistent with this hypothesis is our finding that feeding rats with a diet containing 1% orotic acid for 10 weeks resulted in a liver DNA damage as monitored by its slower sedimentation in alkaline sucrose gradients compared to the corresponding controls. To assess the general applicability of this hypothesis, nucleotide pool imbalances were created by using methods other than feeding orotic acid and their effect on the incidence of γ-glutamyltransferase positive foci in carcinogen initiated rats was determined. The results obtained indicated that rats initiated with 1,2-dimethylhydrazine.2HCl (100 mg/kg) given 18 hr after partial hepatectomy and exposed to diet deficient in arginine, a regimen that causes an increased synthesis and excretion of orotic acid, or were fed diets containing 1% thymidine or 1% thymine developed greater number of γ-glutamyltransferase positive foci compared to the corresponding controls fed the basal diets. These results were interpreted to indicate that orotic acid exerts its promotional effect probably by creating an imbalance in nucleotide pools. One of the mechanisms by which an imbalance of nucleotide pools influences the pathogenesis of the carcinogenic process may be by inducing perturbations in the DNA.
Further Experiments on Lipid Peroxidation in Transplanted and Experimental Hepatomas
Authors: Dianzani Mario U. , Canuto Rosa A. , Rossi Maria A. , Poli Giuseppe , Garcea Renato , Biocca Maria E. , Cecchini Giovanni , Biasi Fiorella , Ferro Margherita , Bassi Anna M.
Published: September 2009

The results of experiments on the subject of lipid peroxidation in hepatomas are described. It is now clear that lipid peroxidation is strongly decreased in most highly dedifferentiated hepatomas. It seems evident that the extent of the decline is strictly related to the degree of dedifferentiation. The model of diethylnitrosamine carcinogenesis, according to the method by Soh, Medline and Farber, has been now adopted to study the stages of carcinogenesis. It was shown that a net decline in lipid peroxidation occurs as early as at the stage of reversible nodules and progresses until the development of clear hepatomas. This change is practically simultaneous with a decline in the efficiency of the enzymes of the drug metabolizing system and in the content of cytochrome P450. Glutathione content and metabolism show also important changes. In fact, a dramatic increase in gamma-glutamyl-transpeptidase takes place very early during carcinogenesis, and is responsible for large decline in total glutathione during incubation of the homogenates. Glutathione peroxidase activity, on the contrary, is decreased, whereas glutathione reductase does not show significant changes. The supernatant of highly anaplastic tumors inhibits lipid peroxidation in normal liver homogenates, suggesting the presence of substances provided with antioxidant properties. These cannot be, however, related to a higher glutathione content. Supernatants from early nodules seem to be unable to block lipid peroxidation in normal liver homogenates. Preliminary experiments done to study the aldehyde pattern produced during lipid peroxidation, both in hepatomas and in nodules, confirm the presence of very poor lipid peroxidation and possibly of different peroxidation kinetics.
Transplantation Studies on Induced and Spontaneous Nodules from B6C3F1Mouse Liver
Authors: Essigmann Ellen , McConnell Robert G. , Newberne Paul M.
Published: September 2009

The present research was designed to assess the potential for progressive growth of hyperplastic, adenomatous, and carcinomatous mouse liver lesions grafted into syngeneic hosts and to determine the effect of treatment type, donor age, and metastatic potential on this parameter. Lesions were obtained from male B6/C3F1 mice fed on an open formula (NIH-007) diet or the same diet mixed with a chlorinated hydrocarbon pesticide (3,5-dichloro-( N-1,1-di-methyl-2-propyl)benzamide) (DcB). Animals were killed at 18 or 24 months of age. Hosts for transplanting were killed at 8 months, or when grafts reached 2 cm in diameter. Lesions diagnosed as nonmalignant (hyperplastic or benign) were more likely to grow after transplantation if obtained after 24 months, rather than after 18 months (0.025 p < 0.05). There was a close correlation between donor lesion morphology and ability to grow in a recipient mouse ( p < 0.0005; 36% of the hyperplastic, 32% of the adenomatous and 79%, of the malignant lesions grew). The majority of lesions retained the morphologic appearance of the donor lesions. Transplanted lesions from control mice were more likely to grow than were those from DCB-treated mice (0.01 < p < 0.025). This correlation was particularly strong for hyperplastic nodules. Metastatic capability in the donor animals correlated positively with both growth potential ( p < 0.01) and growth rate ( p < 0.05) in recipient mice. These findings suggest that progression toward autonomous growth may occur both within the original host and during a prolonged transplantation period, making data interpretation difficult. Lesion morphology alone may not be adequate for assessing capacity for autonomous growth. Other factors, such as evidence of metastasis or local invasion in the primary host, may be required to make more meaningful diagnosis.
Immersion Fixation Methods for Glycol Methacrylate-embedded Testes
Authors: Chapin Robert E. , Ross Monica D. , Lamb James C.
Published: September 2009

Routine processing of testicular tissue through 10% neutral buffered formalin (NBF) into paraffin produces severe cellular shrinkage which obscures most of the morphologic detail. The following studies were performed to compare different combinations of immersion fixatives and embedding media for optimal cellular detail in the final histologic sections. We examined sections of testes from rats, mice, and rabbits fixed in either NBF, Bouin's, Zenker's, or Helly's fixatives, and embedded in either standard paraffin or glycol methacrylate. The results were similar in all 3 species. In paraffin, Bouin's or Helly's fixatives produced the fewest artifacts, while in glycol methacrylate, NBF-fixed tissue showed the greatest intracellular detail and preservation. The merits and limitations of each method are discussed for the rat, with exceptions for the other species noted where appropriate. The use of glycol methacrylate as the support medium for sectioning makes high-quality tissue sections available from formalin-fixed testes.
Lipid Peroxidation and Lipid Antioxidants in Normal and Tumor Cells
Authors: Cheeseman Kevin H. , Burton Graham W. , Ingold Keith U. , Slater Trevor F.
Published: September 2009

Lipid peroxidation is of ten low in tumor tissue as compared to the corresponding normal tissue and it has been postulated that lipid peroxidation may be associated with cell division. In this paper the various contributory factors which control the rate of microsomal lipid peroxidation in normal rat liver and in the Novikoff hepatoma have been carefully analyzed. The low rate of lipid peroxidation in the hepatoma seems to be due to a combination of factors: low levels of polyunsaturated fatty acids and of cytochrome P-450 and elevated levels of lipid-soluble antioxidant. This lipid-soluble antioxidant is principally α-tocopherol.
Quantitative Correlation with Carcinogenic Potency of Different Short Term Tests
Authors: Parodi Silvio , Taningher Maurizio , Boero Paolo , Santi Leonardo
Published: September 2009

The following short term parameters evaluating essentially genotoxic effects were considered: liver DNA alkaline fragmentation (DFI), morphological transformation in hamster embryo cells (TPI), and mutagenicity in the Ames’ test (MPI). The internal consistency of the carcinogenicity data (OPI) was rather high (r = 0.8). The correlation with OPI of DFI and MPI was statistically significant but rather modest, about 0.4-0.5. The best correlation between OPI and TPI was 0.65. This level of correlation was observed only when some kind of dose-response relationship for transformation could be established. When comparisons were attempted for exactly the same 27 compounds for all three tests, a general decrease in predictability was observed. This could be mainly due to problems of representation of the ideal population of chemicals using small samples. The more general problem of the quantitative approach to the predictability of short term tests was also discussed.
Genetic and Environmental Regulation of Aryl Hydrocarbon Hydroxylase in Man: Studies with Liver, Lung, Placenta, and Lymphocytes
Authors: Pelkonen Olavi , Vähäkangas Kirsi , Kärki Niilo T. , Sotaniemi Eero A.
Published: September 2009

Properties and response smoking of aryl hydrocarbon hydroxylase (AHH) activity in various human tissues are reviewed. In the placenta, induction of AHH by smoking can be demonstrated unequivocally. AHH activity in lung samples is variable, but the relation to current or past smoking is unclear. The effect of cigarette smoking can be readily shown in the rate of antipyrine elimination, although there is no change in AHH activity in liver biopsy samples. The reason for this discrepancy is not known. In peripheral lymphocytes a sort of “memory-effect” of cigarette smoking is retained even after culturing, the nature of this phenomenon remaining unclear. Furthermore, there seem to be many factors affecting AHH induction in peripheral lymphocytes in culture. These data suggest that regulation of AHH activity and induction is tissue-specific, i.e. no systemic regulation is discernible. It is also possible that the P-450 isozyme composition in some tissues masks the induction.

Reasons for discrepancies between animal and human data are not clear; however, genuine biological differences and technical difficulties in human studies can be postulated.
Modulatory Mechanisms of Chemical Carcinogenesis: The Role of the NADPH Pool in the Benzo(a)pyrene Activation
Authors: Feo Francesco , Pirisi Lucia , Pascale Rosa , Daino Lucia , Frassetto Serenella , Zanetti Stefania , Garcea Renato
Published: September 2009

Human lymphocytes and human skin fibroblasts isolated in vitro from subjects carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD) exhibit an 86-87% decrease of this enzymatic activity. This is coupled with 51% and 61% decreases of the NADPH/NADP+ ratio in the G6PD-deficient human lymphocytes (HL) and human skin fibroblasts (HSF), respectively. There also occurs a 63-67% decrease of the hexose monophosphate shunt (HMS) in the deficient cells. Incubation with 0.1 mM methylene blue stimulates the HMS of normal HL 15-fold and that of deficient lymphocytes only 2.4-fold. These figures are, respectively, 7 and 2.2 in the case of HSF. This behavior of G6PD-deficient HL and HSF is coupled with an increase of the resistance to the cell death induced by benzo(a)pyrene (BP). This effect is mimicked by the incubation of normal HSF with dehydroepiandrosterone (DEA) which strongly inhibits G6PD. In contrast, no differences between normal and deficient HSF occur as a result of the effect of methylnitrosourea (MNU), a carcinogen that does not need metabolic activation. The NADPH-cytochrome c (P450) reductase of G6PD-deficient HL and HSF homogenates becomes lower than that of controls when endogenous G6PD and exogenous glucose 6-phosphate (G6P) and NADP+ are used as a hydrogen donor system in place of NADPH. Normal and G6PD-deficient HL, having comparable BP-hydroxylating activities, in the presence of exogenous G6P, NADP+, and G6PD, were studied to determine the effect of the absence of exogenous G6PD in the reaction system. When exogenous G6PD is omitted, the deficient HL exhibit a BP hydroxylase activity sharply lower than controls. G6PD-deficient HL and HSF are less prone than controls to produce BP water-soluble metabolites. A decrease in the synthesis of BP metabolites, mutagenic for his– Salmonella typhimurium in G6PD-deficient HL, has also been observed.

These results suggest that the decreased susceptibility of G6PD-deficient cells and HSF to the toxic effect of BP and that of deficient HSF to its previously observed transforming effect, could depend on a reduced BP metabolism in the deficient cells.
Enhancement of Induced Sister Chromatid Exchange and Chromosomal Aberrations by Inhibitors of DNA Repair Processes
Authors: Palitti Fabrizio , Tanzarella Caterina , Degrassi Francesca , DeSalvia Rosella , Fiore Mario
Published: September 2009

The effect of post-treatment with inhibitors of DNA synthesis (hydroxyurea, aphidicolin) and repair (caffeine, 3-aminobenzamide) on the frequencies of chromosomal aberrations and sister chromatid exchange (SCE) induced by mitomycin C and decarbamoyl mitomycin C both in Chinese hamster cells and in human lymphocytes in vitro has been studied. The data show that in the case of Chinese hamster and human lymphocytes mitomycin C-treated cells there is an increased frequency of both chromosomal aberrations and SCE after a G2 post-treatment with the inhibitors, while no increase is observed for decarbamoyl mitomycin C-treated cells. Since SCE are DNA synthesis-dependent, phenomenon, an increase in the frequency of SCE also in the G2 phase might suggest that after mitomycin C treatment there is a residual DNA synthesis still going on very late in the cell cycle.
The Usefulness of Viscometric Analysis for Detecting Minimal Levels of DNA Fragmentation
Authors: Brambilla Giovanni , Carlo Pia , Marinari Umberto
Published: September 2009

Basic requirements for a method used to study the genotoxic potential of xenobiotic agents in the intact animal are sensitivity and capability of detecting the effect of the compound under test in various tissues. A new viscometric technique, which has been found capable of measuring DNA damage in liver, kidney, and lung of rats treated with small single doses of 10 chemical carcinogens, seems to possess such requirements. Single-strand breaks and probably other types of lesions indeed cause changes in DNA supercoiling which can be sensitively measured by monitoring time-dependent changes of DNA viscosity. The main advantage of this technique is that clear-cut modifications of viscometric parameters can be obtained with doses of various carcinogens markedly lower than those found to be the minimal effective ones in other commonly employed short-term in vivo tests. The importance of studying the genotoxic effects of pharmacologically meaningful doses is discussed.
Detoxification of Genotoxic Compounds as a Threshold Mechanism Limiting Their Carcinogenicity
Authors: De Flora Silvio
Published: September 2009

In vitro metabolic trends were assessed for 100 genotoxic agents detected in two bacterial test systems, i.e., the Ames reversion test and a liquid DNA-repair test in Escherichia coli. Seventy-five compounds were found to undergo a more or less pronounced decrease of genotoxicity, in at least one of these models, in the presence of rat liver homogenates or in other metabolic systems (up to 40 different preparations, from various sources, for chromium compounds). A number of these deactivable compounds are reported in the literature to yield negative or equivocal results in animal carcinogenicity assays, which may imply the existence of metabolically regulated thresholds in the initiation of cancer. Several examples are provided to support this hypothesis.

The in vitro treatment with a pharmacologic agent ( N-acetylcysteine) enhanced detoxification mechanisms, either by stimulating enzyme activities promoting glutathione formation in liver homogenates or by reacting itself with direct-acting mutagens and with the genotoxic metabolites of procarcinogens.
Glutathione S-Transferases in Chemically Induced Tumors
Authors: Kraus Pavel , Schulte-Hermann Rolf , Timmermann-Trosiener Irene , Schuppler Jöurg
Published: September 2009

Rats were treated with N-nitrosomorpholin and subsequently with phenobarbital. After several weeks of phenobarbital treatment, foci of altered cells and tumors developed in the livers of the animals. In liver tumors, the activity of glutathione S-transferase was higher than in the surrounding tissue and remained elevated even after the withdrawal of the promoter. Like the glutathio-one S-transferase from normal liver, tumor glutathione S-transferase activity is composed of several, predominantly basic, isoenzymes with an apparent molecular weight of approximately 45,000.
Roles of Gangliosides in the Surface Properties of Normal and Malignant Cells
Authors: Mugnai Gabriele , Tombaccini Donatella , Ruggieri Salvatore
Published: September 2009

Gangliosides are ubiquitous plasma membrane components whose structural characteristics make it possible to establish multiple interactions with the pericellular microenvironment. Several receptorial activities of gangliosides have already been recognized and the possible involvement of gangliosides in growth control, adhesion, differentiation, and immunologic recognition is at present under study. The study of the role of gangliosides in cell growth control has been approached in our laboratory by investigating whether the ganglioside changes found in SV40-transformed Balb/c3T3 cells (SV3T3 cells) revert to a normal pattern in a variant of SV3T3 cells with growth properties similar to those of normal parental cells, the concanavalin A-selected SV3T3 revertant cells. These latter cells showed the same reduction of the more complex gangliosides as found in SV3T3 cells, while their amount of II3NeuAC-LacCer (GM3) was greatly increased compared to that found in either normal or transformed 3T3 cells. In order to study the role of gangliosides in the adhesion process, we analyzed the ganglioside structure of the so-called substrate-attached material, a cell surface structure involved in the adhesion of cells to substrate, from cultures of Balb/c3T3, SV3T3, and concanavalin A-selected SV3T3 revertant cells and from cultures of a system of transformed cells with different metastatic potential: the nonmetastatic B77-3T3 and the highly metastatic AA6 cells. Compared to normal cells, all the transformed cells contained smaller quantities of gangliosides in their substrate-attached material. In both normal and transformed cell lines, the gangliosides of the substrate-attached material, compared to those of detached cells, were found to be enriched by IV3NeuAC, II3NeuAc-GgOse4Cer (GD1a); the only exception were the AA6 cells which contained rather low levels of GD1a in both detached cells and substrate-attached material. When ganglioside compositions of the substrate-attached material, detached cells, and plasma membranes were compared in Balb/c3T3, SV3T3, and concanavalin A-selected SV3T3 revertant cells, a prevalence of GD1a was noted in the substrate-attached material and not in the plasma membranes. Moreover, the level of GD1a was scarcely modified in the substrate-attached material from cultures of a neuroblastoma cell line after growth in media containing chelating agents, a treatment which is known to modify the composition of cellular gangliosides.

The overall indications emerging from these findings suggest a role for GM3 in growth control and a role for complex gangliosides in the adhesion process.
Short- and Long-term Studies on Chemical Carcinogenesis in BALB/Mo Mice
Authors: Chieco-Bianchi Luigi , Aldovini Anna , Ronchese Franca , De Rossi Anita , Majone Franca , Montaldi A. , Levis A. G.
Published: September 2009

To study the interactions between chemical carcinogens and oncogenic retroviruses, BALB/Mo mice which carry the Moloney murine leukemia virus (M-MuLV) as an endogenous virus, and conventional (M-MuLV-free) BALB/c mice, as well as their Bc1 (M-MuLV+ or M-MuLV−) hybrids were injected neonatally with a single dose of urethane. BALB/Mo and V+ Bc1 mice showed accelerated lymphoma development; similar results were obtained in BALB/Mo mice receiving one or two doses of urethane transplacentally. Lung adenomas developed with shorter latency and higher incidence in BALB/Mo mice given urethane at birth; however, significant differences in the incidence of lung adenomas in BALB/Mo mice were found only in two experiments.

Additional short-term experiments were carried out to investigate the mechanism of the higher susceptibility to sister chromatid exchange induction observed in BALB/Mo lymphocytes. It was found that BALB/Mo spleen lymphocytes incubated with cordycepin, an antiviral antibiotic, with or without mitomycin C treatment, showed reduction in both M-MuLV synthesis and sister chromatid exchange frequency, and the latter values were similar to those seen in control cultures.

These data suggest that the integration of M-MuLV proviral DNA into the host genome is per se not sufficient to increase the susceptibility to carcinogenic stimuli, but that other events, such as viral gene expression and amplification, are most likely required for the chemical-viral synergistic effect to occur.
Metabolic Aspects of Cell Cycle Regulation in Normal and Cancer Cells
Authors: Olivotto Massimo , Arcangeli Annarosa , Caldini Riccardo , Chevanne Marta , Cipolleschi Maria G. , Sbarba Persio Dello
Published: September 2009

Several studies are reviewed dealing with the mechanisms which regulate the cell cycle progression in normal and cancer cells. Using Yoshida AH 130 ascites tumor cells, it has been found that the G1-S transition of these cells is impaired by specific inhibitors of the electron flow through the respiratory chain (antimycin A), although respiratory ATP can be replaced by glycolytic ATP. The above transition can be also inhibited by the addition of physiologic substrates, mainly pyruvate, by a mechanism which appears linked to a modification of the cellular redox state and can be totally reversed by adding adenine to the culture medium. Adenine equally removes the block produced by antimycin A, pointing out a respiration-linked step of purine metabolism restricting the cell recruitment into S. A substantial protection of this step against the inhibitory effects of pyruvate and antimycin A has been obtained by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked limiting step of tumor cell cycling involves folate metabolism and its connection to purine synthesis. The biologic relevance of these findings is stressed by the fact that pyruvate addition also inhibits the proliferation of concanavalin A-stimulated lymphocytes as well as of bone marrow hemopoietic cells in the presence of colony-stimulating factors. On the other hand, pyruvate only slightly affects the growth kinetics of malignant lymphoblasts and of Friend erythroleukemia cells either in the absence or in the presence of the differentiation inducer dimethylsulfoxide.
Studies on Chemically Induced Neoplastic Transformation and Mutation in the BALB/3T3 Cl A31-1-1 Cell Line in Relation to the Quantitative Evaluation of Carcinogens
Authors: Saffiotti Umberto , Bignami Margherita , Bertolero Federico , Cortesi Enrico , Ficorella Corrado , Kaighn M. Edward
Published: September 2009

Mutagenesis and neoplastic transformation assays on mammalian cells in culture have been extensively used for quantitative estimates of the activity of carcinogens, in spite of the limitations that such in vitro systems have when compared with in vivo systems for tumor induction. In order to assess the validity of these correlations, a series of studies was undertaken in our laboratory with the BALB/3T3 Cl A31-1-1 mouse embryo cell line. Different carcinogens were found to induce dose-dependent frequencies of transformation, including the direct-acting alkylating agent N-methyI- N'-nitro- N-nitrosoguanidine (MNNG) and carcinogens that were metabolically activated by these cells through different pathways (benzo[a]pyrene, 3-methylcholanthrene, aflatoxin B1, and benzidine). Their respective level of activity on a molar basis was different from that obtained in standard Salmonella + S9 mutagenesis tests. Studies currently underway indicate the possibility of lowering the serum content in the medium considerably, thereby reducing a major variable in the assay. Methods were established for the induction of ouabain-resistant (ouar) mutants in these cells. Studies were conducted by applying 30-min MNNG exposures to cells that were synchronized by serum deprivation followed by serum-induced release from growth block. While maximal induction of mutants occurred in the S phase, the transformation frequency remained constant for treatments in G1 and early or late S. In subsequent studies, cytotoxicity, alkali-labile DNA lesions, ouar mutations, and neoplastic transformation were analyzed concurrently in this cell line after cells were exposed to two concentrations of MNNG and the exposures were protracted for different time periods (30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). A marked temporal dissociation was found in the exposure times required to induce maximal frequencies of mutations and of transformation. Cytotoxicity increased for periods up to 100-200 min; mutations reached a maximal induction level after a much shorter exposure time (30-60 min); DNA damage detected by alkaline elution was already maximal by 30 min. Transformation frequencies, however, reached maximal levels only after exposure periods 1-3 hr longer than those required for maximal mutation. The ratio of transformation to ouar mutation frequencies was 3.7 for short treatment times (30-60 min), but it increased to more than 20 for exposure times of 240 min or longer. These studies support the hypothesis that a single gene mutational event is not sufficient to account for the expression of neoplastic transformation.