Peter Greaves

The rice bran constituent tricin potently inhibits cyclooxygenase enzymes and interferes with intestinal carcinogenesis in ApcMin mice

While brown rice is a staple dietary constituent in Asia, rice consumed in the Western world is generally white, obtained from brown rice by removal of the bran. Rice bran contains the flavone tricin, which has been shown to inhibit colon cancer cell growth. We tested the hypothesis that tricin interferes with adenoma formation in the ApcMin mouse. Mice received tricin (0.2%) in their American Institute of Nutrition 93G diet throughout their postweaning life span (4–18 weeks). Consumption of tricin reduced numbers of intestinal adenomas by 33% (P < 0.05) compared with mice on control diet. We explored whether tricin may exert its effect via inhibition of cyclooxygenase (COX) enzymes. Its effect on COX activity was assessed in purified enzyme preparations in vitro and its ability to reduce prostaglandin E2 (PGE2) levels in human colon–derived human colon epithelial cell (HCEC) and HCA-7 cells in vitro and in ApcMin mice in vivo. Tricin inhibited activity of purified COX-1 and COX-2 enzyme preparations with IC50 values of ∼1 μmol/L. At 5 μmol/L, it reduced PGE2 production in HCEC or HCA-7 cells by 36% (P < 0.01) and 35% (P < 0.05), respectively. COX-2 expression was reduced by tricin weakly in HCEC and unaffected in HCA-7 cells. PGE2 levels in the small intestinal mucosa and blood of ApcMin mice that had received tricin were reduced by 34% (P < 0.01) and 40% (P < 0.05), respectively, compared with control mice. The results suggest that tricin should be further evaluated as a putative colorectal cancer chemopreventive agent.

Cancer chemoprevention: Evidence of a nonlinear dose response for the protective effects of resveratrol in humans and mice

Low-dose resveratrol prevents tumor growth in mice and in human tissues, suggesting a revision of development strategies for preventive dietary agents. Less is more From energy drinks to supplements to skin serums, resveratrol has been sold to the public for myriad health benefits, most famously in the anti-aging arena. In fact, at a posh wine bar, one might overhear a patron lamenting the small dose of resveratrol one receives in a glass of the red variety. Now, Cai et al. show that a low rather than a high dose of resveratrol prevents tumor growth in mice and alters metabolic pathways in human tissues. The authors compared the dose-response curves of a dietary dose of resveratrol and a 200-fold higher amount in mice that spontaneously develop colorectal adenomas—precursors to cancer—that were fed a standard or a high-fat diet. In the mice on the high-fat diet, low-dose resveratrol reduced intestinal tumor development much better than did the high dose. In mouse tumor cells, resveratrol efficacy was tracked with an increase in autophagy and senescence markers and activation of adenosine monophosphate (AMP)–activated protein kinase (AMPK)—an enzyme that functions in the maintenance of cellular energy homeostasis. Exposure of human colorectal cancer tissue to low concentrations of resveratrol also caused an increase in autophagy and activation of AMPK. Colorectal mucosal samples isolated from cancer patients who received a low-dose resveratrol regimen before tumor resection showed an increase in expression of the cytoprotective, oxidative stress-activated enzyme NAD(P)H dehydrogenase, quinone 1 (NQO1). These findings suggest that resveratrol operates by modulating energy balance and responding to stress. At a time when “supersizing” is popular, the nonlinear dose-response documented in the new work suggests that its time for a revision in development strategies for preventative dietary agents. Resveratrol is widely promoted as a potential cancer chemopreventive agent, but a lack of information on the optimal dose prohibits rationally designed trials to assess efficacy. To challenge the assumption that “more is better,” we compared the pharmacokinetics and activity of a dietary dose with an intake 200 times higher. The dose-response relationship for concentrations generated and the metabolite profile of [14C]-resveratrol in colorectal tissue of cancer patients helped us to define clinically achievable levels. In ApcMin mice (a model of colorectal carcinogenesis) that received a high-fat diet, the low resveratrol dose suppressed intestinal adenoma development more potently than did the higher dose. Efficacy correlated with activation of adenosine monophosphate–activated protein kinase (AMPK) and increased expression of the senescence marker p21. Nonlinear dose responses were observed for AMPK and mechanistic target of rapamycin (mTOR) signaling in mouse adenoma cells, culminating in autophagy and senescence. In human colorectal tissues exposed to low dietary concentrations of resveratrol ex vivo, we measured enhanced AMPK phosphorylation and autophagy. The expression of the cytoprotective NAD(P)H dehydrogenase, quinone 1 (NQO1) enzyme was also increased in tissues from cancer patients participating in our [14C]-resveratrol trial. These findings warrant a revision of developmental strategies for diet-derived agents designed to achieve cancer chemoprevention.

Male Genital Tract

During the course of drug development, the effects of novel compounds on the reproductive system are examined in specialized reproductive tests. However, these studies are infrequently conducted before the first dose of a novel pharmaceutical to humans. Hence, the only assessment of the male reproductive organs performed prior to the first administration of a novel therapeutic agent to man is histological examination of the male sex organs in conventional toxicity studies in rodent and nonrodent species. For this reason, this assessment should be performed using meticulous techniques. This approach has been agreed in the consensus guidelines from the International Conference on Harmonisation following the studies that showed histopathological examination of the male reproductive organs in the rodent 4-week toxicity study was more sensitive in detecting effects than fertility studies. In mammals the male sex organs comprise the paired testes and the accessory reproductive organs: prostate, seminal vesicles, coagulating glands, ampullary glands, and epididymis. Cytochemical or immunocytochemical demonstration of acid phosphatase has been shown to be a good indicator of the functional integrity of prostatic secretory cells, particularly as acid phosphatase is a major secretory component, which is androgen-dependent in man and laboratory animals. It has been shown that there is a close immunological cross-reactivity of antibody against acid phosphatase from the rat ventral prostate, the canine, and human prostate.

Liver and Pancreas

The purpose of this chapter is to discuss the importance of the pathologic alterations that can be induced by drugs in the liver of laboratory animals and their implication for hepatic toxicity in patients. It describes anatomy and physiology of the liver of the laboratory species in some detail and discusses how these may differ from humans. Drug-induced hepatic alterations described include changes to hepatic weight, hypertrophy, hyperplasia, storage disorders, pathology of the bile duct and gallbladder as well as the frequently occurring neoplasms in the rodent liver and their implications for human therapy. The chapter also describes a similar range of changes that can be seen in the exocrine pancreas and the endocrine alterations in the islets of Langerhans.

Histopathology Of Hemangiosarcomas in Mice and Hamsters and Liposarcomas/Fibrosarcomas in Rats Associated with PPAR Agonists

Peroxisome proliferator-activated receptors (PPAR) are involved in the pathogenesis of insulin resistance, diabetes, and related complications. Consequently, the identification of PPAR subtypes and the potential for their activation provides promising therapeutic targets for the management of type 2 diabetes mellitus. Available data from rodent carcinogenicity studies, however, demonstrate that PPAR agonists can be tumorigenic in one or more species of rodents at multiple sites. In 2005, the Health and Environmental Sciences Institute (HESI) PPAR Agonist Project Committee was established by a group of pharmaceutical companies to advance research on and to understand the modes of action and human relevance of this emerging rodent tumor data for PPAR agonists. Since the most commonly observed tumor types reported in rodents are hemangiosarcomas, fibrosarcomas and liposarcomas, the PPAR Agonist Project Committee approved a Pathology Working Group (PWG) to develop consensus of morphologic criteria for tumor diagnoses and consistency of diagnoses across multiple studies for hemangiosarcomas in mice and hamsters and liposarcomas/fibrosarcomas in rats. Therefore, the focus of the PWG review was to establish consistent tumor diagnostic criteria, to assess evidence of potentially preneoplastic changes and to identify distinguishing morphologic differences which may exist between spontaneous changes present in control animals with similar changes from treated animals. Specific diagnostic criteria and nomenclature are recommended for the classification of proliferative vascular lesions which may be present in mice or hamsters and for proliferative mesenchymal changes in rats in studies that are conducted with PPAR agonists.

Patterns of drug-induced cardiovascular pathology in the beagle dog: relevance for humans

In toxicity studies, the examination of tissue sections for pathological changes is the principle method for the identification of organ toxicity and characterisation of the hazard of novel drugs for humans. Study of the patterns of pathological alterations also represents an important means of developing an understanding of the mechanism of toxicity. However as pathological change frequently represents a final common expression of diverse processes, additional functional information is often required for a clear understanding of the mechanisms of toxicity. This is exemplified in the evaluation of the effects of drugs on the beagle dog cardiovascular system where an understanding of mechanisms is crucial in the assessment of human risk. Particular patterns of drug-induced structural change in the myocardium or blood vessels are frequently linked to specific mechanisms of toxicity. However, assessment based on the interpretation of patterns of cardiovascular pathology alone may be misleading. Quite different changes in cardiac and vascular function or direct cellular toxicity may also be manifest by pathological features in common. Therefore, a clear understanding of mechanism frequently requires additional in vivo or in vitro physiological, pharmacological, biochemical or other mechanistic information. The beagle dog remains an important model for the study of cardiovascular toxicity because in this species, haemodynamic changes and pathological alterations can be related in a way that provides the basis for the safe study in humans of novel drugs with cardiovascular activity.

Metabolic profiling of transgenic adenocarcinoma of mouse prostate (TRAMP) Tissue by 1H‐NMR analysis: evidence for unusual phospholipid metabolism

The TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse model has frequently been used in preclinical studies with chemotherapeutic/chemopreventive rationales. Here the hypothesis was tested using 1H‐NMR‐based metabolic profiling that the TRAMP tumor metabolic phenotype resembles that reported for human prostate cancer.

Evaluation of the cancer chemopreventive efficacy of silibinin in genetic mouse models of prostate and intestinal carcinogenesis: Relationship with silibinin levels

Silibinin, a flavonolignan from milk thistle seeds, possesses cancer chemopreventive properties in rodent models of carcinogenesis. We tested the hypotheses that silibinin or silipide, silibinin formulated with phospholipids, delays tumour development in TRAMP or Apc(Min) mice, genetic models of prostate or intestinal malignancies, respectively. Mice received silibinin or silipide with their diet (0.2% silibinin equivalents) from weaning. Intervention with silipide reduced the size of well differentiated TRAMP adenocarcinomas by 31%. Silipide and silibinin decreased the incidence of poorly differentiated carcinomas by 61% compared to mice on control diet. Silipide decreased plasma levels of insulin-like growth factor (IGF)-1 by 36%. Levels of circulating IGF binding protein (IGFBP)-3 in mice on silipide or silibinin were 3.9- or 5.9-fold, respectively, elevated over those in control TRAMP mice. In Apc(Min) mice silibinin, but not silipide, had only a marginal adenoma number-reducing effect. The results cautiously support the advancement of silipide to the stage of clinical investigation in prostate cancer.

Proliferative and Non-Proliferative Lesions of the Rat and Mouse Soft Tissue, Skeletal Muscle and Mesothelium

Abstract The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the soft tissues including skeletal muscle as well as the mesothelium of rats and mice. The standardized nomenclature of lesions presented in this document is also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions in soft tissues, skeletal muscle and mesothelium in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. (DOI: 10.1293/tox.26.1S; J Toxicol Pathol 2013; 26: 1S–26S)

A GC–MS metabolic profiling study of plasma samples from mice on low- and high-fat diets☆

Metabolic profiling of biofluids, based on the quantitative analysis of the concentration profile of their free low molecular mass metabolites, has been playing increasing role employed as a means to gain understanding of the progression of metabolic disorders, including obesity. Chromatographic methods coupled with mass spectrometry have been established as a strategy for metabolic profiling. Among these, GC-MS, targeting mainly the primary metabolism intermediates, offers high sensitivity, good peak resolution and extensive databases. However, the derivatization step required for many involatile metabolites necessitates specific data validation, normalization and analysis protocols to ensure accurate and reproducible performance. In this study, the GC-MS metabolic profiles of plasma samples from mice maintained on 12- or 15-month long low (10 kcal%) or high (60 kcal%) fat diets were obtained. The profiles of the trimethylsilyl(TMS)-methoxime(MeOx) derivatives of the free polar metabolites were acquired through GC-(ion trap)MS, using [U-(13)C]-glucose as the internal standard. After the application of a recently developed data correction and normalization/filtering protocol for GC-MS metabolomic datasets, the profiles of 48 out of the 77 detected metabolites were used in multivariate statistical analysis. Data mining suggested a decrease in the activity of the energy metabolism with age. In addition, the metabolic profiles indicated the presence of subpopulations with different physiology within the high- and low-fat diet mice, which correlated well with the difference in body weight among the animals and current knowledge about hyperglycemic conditions.