Of Mice and Women: Light as a Circadian Stimulus in Breast Cancer Research

OBJECTIVE: Nocturnal rodents are frequently used as models in human breast cancer research, but these species have very different visual and circadian systems and, therefore, very different responses to optical radiation or, informally, light. Because of the impact of light on the circadian system and because recent evidence suggests that cancer risk might be related to circadian disruption, it is becoming increasingly clear that optical radiation must be properly characterized for both nocturnal rodents and diurnal humans to make significant progress in unraveling links between circadian disruption and breast cancer. In this paper, we propose a quantitative framework for comparing radiometric and photometric quantities in human and rodent studies. METHODS: We reviewed published research on light as a circadian stimulus for humans and rodents. Both suppression of nocturnal melatonin and phase shifting were examined as outcome measures for the circadian system. RESULTS: The data were used to develop quantitative comparisons regarding the absolute and spectral sensitivity for the circadian systems of humans and nocturnal rodents. CONCLUSIONS: Two models of circadian phototransduction, for mouse and humans, have been published providing spectral sensitivities for these two species. Despite some methodological variations among the studies reviewed, the circadian systems of nocturnal rodents are approximately 10,000 times more sensitive to optical radiation than that of humans. Circadian effectiveness of different sources for both humans and nocturnal rodents are offered together with a scale relating their absolute sensitivities. Instruments calibrated in terms of conventional photometric units (e.g., lux) will not accurately characterize the circadian stimulus for either humans or rodents.     

Macromolecular adduct formation and metabolism of heterocyclic amines in humans and rodents at low doses.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are heterocyclic amines formed during the cooking of meat and fish. Both are genotoxic in a number of test systems and are carcinogenic in rats and mice. Human exposure to these compounds via dietary sources has been estimated to be under 1 microg/kg body wt. per day, although most laboratory animal studies have been conducted at doses in excess of 10 mg/kg body wt. per day. We are using accelerator mass spectrometry (AMS), a tool for measuring isotopes with attomole sensitivity, to study the dosimetry of protein and DNA adduct formation by low doses of MeIQx and PhIP in rodents and comparing the adduct levels to those formed in humans. The results of these studies show: 1, protein and DNA adduct levels in rodents are dose-dependent; 2, adduct levels in human tissues and blood are generally greater than in rodents administered equivalent doses; and 3, metabolite profiles differ substantially between humans and rodents for both MeIQx and PhIP, with more N-hydroxylation (bioactivation) and less ring oxidation (detoxification) in humans. These data suggest that rodent models do not accurately represent the human response to heterocyclic amine exposure.     

Mutation of beta-catenin is an early event in chemically induced mouse hepatocellular carcinogenesis.

beta-catenin activation, and subsequent upregulation of Wnt-signaling, is an important event in the development of certain human and rodent cancers. Recently, mutations in the beta-catenin gene in the region of the serine-threonine glycogen kinase (GSK)-3beta phosphorylation target sites have been identified in hepatocellular neoplasms from humans and transgenic mice. In this study we examined 152 hepatocellular neoplasms from B6C3F1 mice included in five chemical treatment groups and controls for mutations in the beta-catenin gene. Twenty of 29 hepatocellular neoplasms from mice treated with methyleugenol had point mutations at codons 32, 33, 34 or 41, sites which are mutated in colon and other cancers. Likewise, nine of 24 methylene chloride-induced hepatocellular neoplasms and 18 of 42 oxazepam-induced neoplasms exhibited similar mutations. In contrast, only three of 18 vinyl carbamate-induced liver tumors, one of 18 TCDD-induced liver tumors, and two of 22 spontaneous liver neoplasms had mutations in beta-catenin. Thus, there appears to be a chemical specific involvement of beta-catenin activation in mouse hepatocellular carcinogenesis. Expression analyses using Western blot and immunohistochemistry indicate that beta-catenin protein accumulates along cell membranes following mutation. The finding of mutations in both adenomas and carcinomas from diverse chemical treatment groups and the immunostaining of beta-catenin protein in an altered hepatocellular focus suggest that these alterations are early events in mouse hepatocellular carcinogenesis.     

Genetic alterations in liver carcinogenesis: implications for new preventive and therapeutic strategies

In this review, genetic changes known to occur in human and experimental animal hepatocarcinogenesis are evaluated comparatively, with the aim of identifying genes that could potentially be targets of new preventive and therapeutic strategies, albeit the fact that although a step-by-step analysis of the premalignant stages has been largely accomplished in experimental hepatocarcinogenesis, this goal is still elusive in the case of humans. Overexpression of several of the genes implicated in the MAPK signaling cascade and cell cycle control appears to be most likely responsible for initiated cells acquiring a proliferating phenotype that facilitates the accumulation of structural changes in additional genes, resulting in the generation of autonomously growing preneoplastic and neoplastic lesions. Several gene abnormalities seen in precancerous lesions of rodents also occur in human hepatocellular carcinomas, suggesting that at least some of them could be present also in human precancerous lesions. Furthermore, there are reports that epigenetic events, such as abnormal DNA methylation, may be critical in hepatocarcinogenesis. DNA hypomethylation is an early event, both in human and experimental hepatocarcinogenesis, and its role in the activation of various genes, has been postulated. In recent years, linkage analysis studies have led to the identification of susceptibility/resistance loci that influence the progression stage of hepatocarcinogenesis in mice and rats. The relevance of these findings, though, will depend on the identification of the genes, and on whether in humans there are genes ortholog with rodent's susceptibility/resistance genes. It is proposed that rodent hepatocarcinogenesis represents a promising model for the identification of genes implicated in the early stages of the process, and that many of these genes may represent key targets for the application of gene therapy in the prevention and treatment of liver cancer.     

Validity and limitations of animal experiments in assessing lung carcinogenicity of cadmium.

Several cadmium compounds have been observed to induce in rats, but in rats only, a dose-dependent increase in lung cancer. A similar sensitivity to lung cancer induction in both humans and rats can be deduced from a comparison of the histogenesis of tumours and the dose response to radiation, since similar numbers of DNA lesions are produced. Since the carcinogenic action of cadmium is limited to the site of deposition, the toxicokinetics of inhaled particles in human and rodents are discussed in relation to the exposure of the respective target cells in both species. It is stressed that the rat may be much more sensitive to the induction of cancer following the retention of poorly soluble compounds. A comparison of the possible dose-effect response in humans and the dose response in rats showed that the shape of the "dose" response cannot be extrapolated. Finally, clonogenicity and DNA repair of tracheal cells sublethally exposed in vitro to cadmium differ significantly in rats and hamsters. This may explain why hamsters exposed in vivo do not develop tumours.     

Are inflammatory cytokines the common link between cancer-associated cachexia and depression?

The prevalence of depression among patients diagnosed with cancer is higher than among the general medical population and is associated with faster tumor progression and shortened survival time. Cancer-related depression often occurs in association with anorexia and cachexia, although until recently the relationship between these conditions has not been well understood. Cachexia is associated with poorer quality of life and survival outcomes and is theeventual cause of death in approximately 30% of all patients with cancer. Recent evidence has linked elevated levels of inflammatory cytokines with both depression and cachexia, and experiments have shown that introducing cytokines induces depression and cachectic symptoms in both humans and rodents, suggesting that there may be a common etiology at the molecular level. Therapeutic agents targeting specific cytokine molecules, such as interleukin-6 or tumor necrosis factor-alpha, are currently being evaluated for their potential to simultaneously treat both depression and cachexia pharmacologically. This review summarizes the available data suggesting a dual role for cytokines in the development of cancer-related depression and cachexia and describes how biologic therapies targeting specific cytokines may improve outcomes beyond depression and cachexia, such as survival and quality of life.     

Rodent Carcinogenicity of Peroxisome Proliferators and Issues on Human Relevance

Abstract

A variety of substances such as hypolipidemic drugs, phthalate ester plasticizers, pesticides, and industrial solvents have been shown to increase the size and number of peroxisomes in rats and mice. They are grouped under the generic term peroxisome proliferators (PP) because of their unique property of inducing peroxisome proliferation. There are marked species differences in response to PP. Rats and mice are most sensitive, and hamsters show an intermediate response while guinea pigs, monkeys, and humans appear to be relatively insensitive or non-responsive at dose levels that produce a marked response in rodents. Out of over 100 PP identified to date, about 30 have been adequately tested and shown to be carcinogenic, inducing tumors (primarily in the liver) upon chronic administration to rats and/or mice; hence, chemicals which induce the proliferation of peroxisomes have formed a unique class of chemical carcinogens. It is now well documented that activation of the “peroxisome proliferator-activated receptor α” (PPARα) is involved in PP-induced liver growth and carcinogenesis in rodents. PPAR,is also present in human cells; however, the levels reported are about 10% of those found in the liver of rodents. The human relevance of rodent tumors induced by PP has been the subject of debate over the last decade. Review of the existing evidence on PPAR-,agonists by a recent International Life Science Institute (ILSI) workgroup following a human relevance mode of action (MOA) framework has concluded that despite the presence of similar pathways in humans, it is unlikely that the proposed MOA for rodent tumors is plausible in humans, taking into account kinetic and dynamic factors. The data, however, did not permit a definitive conclusion that the animal MOA is not plausible in humans. While these agents appear unlikely to be hepatocarcinogens in humans at expected levels of human exposure, it remains uncertain to some experts in the field whether there is no possibility of carcinogenic potential under any circumstance of PP exposure, and if the potential human carcinogenicity of these chemicals can be summarily ignored. A number of remaining issues on human relevance of rodent tumors induced by PP are discussed.     

Comparative functional genomics for identifying models of human cancer

Genetically modified mice with overexpressed and/or deleted genes have been used extensively to model human cancer. However, it is uncertain as to what extent the mouse models reproduce the corresponding cancers in humans. We have compared the global gene expression patterns in human and mouse hepatocellular carcinomas (HCCs) in an attempt to identify the mouse models that most extensively reproduce the molecular pathways in the human tumors. The comparative analysis of the gene expression patterns in murine and human HCC indicates that certain genetic mouse models closely reproduce the gene expression patterns of HCC in humans, while others do not. Identification of mouse models that reproduce the molecular features of specific human cancers (or subclasses of specific human cancers) promises to accelerate both the understanding of the molecular pathogenesis of cancer and the discovery of therapeutic targets. We propose that this method, comparative functional genomics, could be effectively applied to the analysis of mouse models for other human cancers.     

Higher susceptibility to aflatoxin B1‐related hepatocellular carcinoma in glycine N‐methyltransferase knockout mice

In both humans and rodents, males are known to be more susceptible than females to hepatocarcinogenesis. We have previously reported that glycine N‐methyltransferase (GNMT) interacts with aflatoxin B₁ (AFB₁) and reduces both AFB₁‐DNA adduct formation and hepatocellular carcinoma (HCC) in mice. We also reported that 50% of the males and 100% of the females in a small group of Gnmt null (Gnmt?/?) mice developed HCC, with first dysplastic hepatocellular nodules detected at mean ages of 17 and 16.5 months, respectively. In our study, we tested our hypothesis that male and female Gnmt?/? mice are susceptible to AFB₁ carcinogenesis, and that the absence of Gnmt expression may accelerate AFB₁‐induced liver tumorigenesis. We inoculated Gnmt?/? and wild‐type mice intraperitoneally with AFB₁ at 7 days and 9 weeks of age and periodically examined them using ultrasound. Dysplastic hepatocellular nodules were detected in six of eight males and five of five females at 12.7 and 12 months of ages, respectively. Dysplastic hepatocellular nodules from 5/8 (62.5%) male and 4/5 (80%) female Gnmt?/? mice were diagnosed as having HCC, ～6 months earlier than AFB₁‐treated wild‐type mice. Results from microarray and real‐time PCR analyses indicate that five detoxification pathway‐related genes were downregulated in AFB₁‐treated Gnmt?/? mice: Cyp1a2, Cyp3a44, Cyp2d22, Gsta4 and Abca8a. In summary, we observed overall higher susceptibility to AFB₁‐related HCC in Gnmt?/? mice, further evidence that GNMT overexpression is an important contributing factor to liver cancer resistance.     

Sixth aspen cancer conference: Molecular mechanisms of genetic deregulation in toxicity and carcinogenesis

The multidrug transporter P-glycoproteins are encoded by three multidrug-resistance (mdr) genes in rodents, designated mdr1a (mdr3), mdr1b (mdr1), and mdr2. Only the first two genes are functionally related to multidrug resistance. Activation of rodent mdr genes during liver regeneration and hepatocarcinogenesis has been reported. In mice, mdr1a is activated in hepatocellular carcinomas (HCCs) produced by various carcinogenic protocols, whereas both mdr1a and mdr2 are activated during liver regeneration. In this communication, we report isolating three gene-specific probes for the rat mdr homologues, which were used as probes in an RNase protection assay to demonstrate that mdr1b mRNA was expressed in HCCs induced by two different protocols. Furthermore, high levels of hepatic mdr1b mRNA but only moderate levels of mdr1a and mdr2 mRNA were seen in preneoplastic lesions in rats treated with 2-acetylaminofluorene. Likewise, highly elevated levels of hepatic mdr1b mRNA but only moderately increased levels of mdr1a and mdr2 mRNA were seen after partial hepatectomy. Nevertheless, the general patterns of tissue-specific expression of these three mdr genes were similar in rats and mice. These results reveal a complex hepatic gene expression pattern during hepatocarcinogenesis and hepatic proliferation for this conserved gene family in rodents.     

Chemoprevention of cancer--focusing on clinical trials

Chemoprevention of cancer is reviewed from the viewpoints of action mechanisms and methodology of clinical trials in order to introduce promising agents discovered by in vitro and/or in vivo studies to applications in humans. The clinical trial procedure essentially follows the phase study which has been employed for chemotherapeutic drugs. Chemoprevention of bladder cancer, prostate cancer, gastric cancer, hepatocellular carcinoma, breast cancer, head and neck cancer, colorectal cancer and lung cancer is reviewed, mainly focusing on clinical trials. Previous clinical trials have shown the effectiveness of the following: polyprenoic acid (acyclic retinoid) for hepatocellular carcinoma; tamoxifen for breast cancer; retinoic acids for head and neck tumor; and aspirin, a COX-2 inhibitor, for colorectal cancer. Despite the advantageous effects of some of these agents, their toxic effects must also be of concern at the same time. For example, in a chemoprevention trial of lung cancer, beta-carotene was unexpectedly found to increase the risk of lung cancer among high-risk groups. It is also noted that large-scale clinical trials demand large research grants, which may not be affordable in Japan. Chemoprevention is still an emerging field of oncology where researchers in both basic and clinical sciences face great challenges.     

Time-dose factors in radiotherapy: a review of the human data

The values for alpha/beta (fractionation sensitivity, or recovery capacity) for early and late reactions in human normal tissues are consistent with results from experimental animals. For breast treatments direct analysis indicates that for early reactions alpha/beta is in the range 7 to 11 Gy, while for late effects it is in the range 2 to 4 Gy. Data on recovery kinetics in human tissues is limited but these indicate that recovery may be slower in humans than in rodents. For early skin reactions the halftime of recovery is about 1 h, while for late telangiectasia it is more than 3 h. alpha/beta values for human tumors are more variable than in rodents: some are high (head and neck, lung, skin, cervix) and similar to those for early reacting normal tissues. Others are low, including melanomas, where alpha/beta was estimated at 0.6 (-1.1, 2.5) Gy, and liposarcomas, where direct analysis of cases surveyed from the literature suggested that alpha/beta = 0.4 (-1.4, 5.4) Gy. Repopulation kinetics is faster in the mucosa of the soft palate and faucial pillars (1.8 Gy/day) than in head and neck tumors (up to 1 Gy/day).     

Chronic infection with hepatitis G virus in relation to hepatocellular carcinoma among non-Asians in Los Angeles County, California.

BACKGROUND: The recently identified hepatitis G virus (HGV) is a hepatotropic RNA virus belonging to the Flaviviridae family. The virus causes chronic viremia, and exposure to blood products is a recognized route of transmission in humans. To the authors' knowledge there is scant information regarding the hepatocarcinogenic potential of HGV. The current study examined the association between HGV infection and the risk of hepatocellular carcinoma. METHODS: A population-based, case-control study involving 144 non-Asian patients with hepatocellular carcinoma who were ages 18-74 years at diagnosis and 252 community controls of similar age, gender, and race was conducted in Los Angeles, California. Study subjects were assessed for serologic markers of infections with the hepatitis B virus (hepatitis B surface antigen, antibody to the hepatitis B core antigen, and antibody to the hepatitis B surface antigen), hepatitis C virus (HCV) (anti-HCV and HCV RNA), and HGV (HGV RNA). RESULTS: Twelve of the 144 hepatocellular carcinoma patients (8.3%) and 5 of the 252 control subjects (2.0%) were positive for serum HGV RNA. The presence of HGV RNA in the serum was associated with a statistically significant 5.4-fold risk of hepatocellular carcinoma (95% confidence limit, 1.8, 16.6). The excess risk for hepatocellular carcinoma among HGV-infected individuals was independent of the effects of hepatitis B and hepatitis C infections. CONCLUSIONS: Chronic infection with HGV may play a role in the development of hepatocellular carcinoma. If the observed statistical association is a causal one, then infection with HGV may account for approximately 8% of hepatocellular carcinoma cases occurring in non-Asians in Los Angeles, California.     

Hepatocyte-restricted constitutive activation of PPAR alpha induces hepatoproliferation but not hepatocarcinogenesis

Peroxisome proliferator-activated receptor alpha (PPARalpha) is responsible for peroxisome proliferator-induced pleiotropic responses, including the development of hepatocellular carcinoma in rodents. However, it remains to be determined whether activation of PPARalpha only in hepatocytes is sufficient to induce hepatocellular carcinomas. To address this issue, transgenic mice were generated that target constitutively activated PPARalpha specifically to hepatocytes. The transgenic mice exhibited various responses that mimic wild-type mice treated with peroxisome proliferators, including significantly decreased serum fatty acids and marked induction of PPARalpha target genes encoding fatty acid oxidation enzymes, suggesting that the transgene functions in the same manner as peroxisome proliferators to regulate fatty acid metabolism. However, the transgenic mice did not develop hepatocellular carcinomas, even though they exhibited peroxisome proliferation and hepatocyte proliferation, indicating that these events are not sufficient to induce liver cancer. In contrast to the transgenic mice, peroxisome proliferators activate proliferation of hepatic non-parenchymal cells (NPCs). Thus, activation of hepatic NPCs and/or associated molecular events is an important step in peroxisome proliferators-induced hepatocarcinogenesis.     

Analysis of ras gene mutations in human hepatic malignant tumors by polymerase chain reaction and direct sequencing

The ras gene is one of the oncogenes most commonly detected in human cancers, and it consists of three families (H-ras, K-ras, N-ras). These genes are converted to active oncogenes by point mutations occurring in either codon 12, 13, or 61. We analyzed mutations of these codons in 23 primary hepatic malignant tumors (12 hepatocellular carcinomas, nine cholangiocarcinomas, and two hepatoblastomas) by a method to directly sequence nucleotides, using polymerase chain reaction and a direct sequencing method. Of 23 hepatic malignant tumors, point mutations at K-ras codon 12 or K-ras codon 61 were found in six of nine cholangiocarcinomas. In contrast, there were no point mutations in any of 12 hepatocellular carcinomas or two hepatoblastomas around codon 12, 13, or 61 of the ras genes. These observations suggest that ras gene mutations are not related to pathogenesis of hepatocellular carcinoma, but play an important role in pathogenesis of cholangiocarcinoma.     

Human gene therapy: Present and future

Summary The hematopoietic system and the liver are two primary target organs for attempting somatic gene therapy of hereditary deficiencies. Several leading laboratories have recently been able to demonstrate that bone marrow cells from rodents and non-human primates can be successfully transduced with foreign genes, resulting in the functional expression of these genes in culture. The genetically reconstituted cells can subsequently be transplanted into X-irradiated recipients, and expression of the transduced genes is observed in the recipients for more than 6 months. Subsequently, gene transfer into peripheral T-lymphocytes in humans has been attempted, and the clinical trials are currently in progress. The liver is the other major organ under intensive investigation. Primary hepatocytes can be isolated from rodents, rabbits, and dogs, and successfully transduced with recombinant retroviruses. After autologous transplantation, long term survival of the engrafted cells in vivo has been observed. More recently, it has been shown that human hepatocytes can also be efficiently transduced with recombinant retroviruses. These experimental results have laid the foundation for somatic gene therapy of hereditary deficiencies in humans in the future.     

Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts

A number of promutagenic exocyclic DNA adducts have recentlybeen detected in both humans and rodents without carcinogentreatment These observations raised questions about their originsand potential significance in carcinogenesis. In this commentary,we present our views pertaining to the in vivo sources of thesecyclic adducts, specifically the cyclic propano and etheno adducts.The basis for our discussion comes mainly from the informationgenerated through a span of more than a decade from severallaboratories, including ours. This commentary summarizes thedata from the chemical and biochemical studies that providesupport for the hypothesis that lipid peroxida-tion is involvedin the endogenous formation of these exocyclic adducts.     

Nonviral risk factors for hepatocellular carcinoma in a low-risk population, the non-Asians of Los Angeles County, California.

We conducted interviews on 74 patients with histologically confirmed hepatocellular carcinoma. These patients, aged 18-74 years, were black or white residents of Los Angeles County. We also interviewed 162 population control subjects who were comparable to the case patients by age, sex, and race. Cigarette smoking was a significant risk factor for hepatocellular carcinoma [relative risk (RR) = 2.1; 95% confidence limits (CL) = 1.1, 4.0]; the effects were similar in men and in women. Heavy alcohol consumption was another risk factor for hepatocellular carcinoma in men; men who consumed 80 g or more of ethanol per day had an RR of 4.7 (95% CL = 1.4, 15.4) relative to those who had never drunk alcohol on a weekly basis. The level of alcohol intake was relatively low in women, and no significant effect on risk of hepatocellular carcinoma was observed. Use of oral contraceptives was significantly related to risk of hepatocellular carcinoma in women (RR = 3.0; 95% CL = 1.0, 8.8); those who were exposed for more than 5 years exhibited a 5.5-fold increased risk (95% CL = 1.2, 24.8). The effects of these three risk factors on hepatocellular carcinoma development were independent of each other and independent of serologically determined viral hepatitis. Our data suggest that cigarette smoking, alcohol consumption, and use of oral contraceptives are major risk factors for hepatocellular carcinoma among non-Asian residents of Los Angeles County. We also observed a significant association between a history of diabetes and hepatocellular carcinoma (RR = 3.3; 95% CL = 1.5, 7.2), especially among those who had received insulin treatment (RR = 18.5; 95% CL = 2.2, 156.0). This association may have etiological significance.