Jeffrey C. Wolf

Qualitative and Quantitative Analysis of Nonneoplastic Lesions in Toxicology Studies

A pathology report is written to convey information concerning the pathologic findings in a study. This type of report must be complete, accurate and communicate the relative importance of various findings in a study. The overall quality of the report is determined by three Quality Indicators: thoroughness, accuracy, and consistency. Thoroughness is the identification of every lesion present in a particular organ or tissue, including spontaneous background lesions. Experienced pathologists familiar with background lesions may disregard certain types of lesions or establish a threshold or a severity above which background lesions are diagnosed. Accuracy is the ability to make, and precisely communicate, correct diagnoses. Nomenclature of lesions is a matter of definition and experienced pathologists generally agree as to what terms are to be used. Consistency is the uniform use of a specific term to record a defined lesion and implies that the same diagnostic criteria are being followed for each type of diagnosis. The relative severity of nonneoplastic lesions can be recorded either semiquantitatively or quantitatively. Semiquantitative analysis involves the application of defined severity grades or ranges for specific lesions. Quantitative analysis (counts and measurements) can be performed manually or electronically, utilizing image analysis and stereological techniques to provide numerical values. When both qualitative and quantitative parameters are applied in preparation of a pathology report, the recorded pathology findings can be interpreted and put into perspective. The use of this approach assures a reader that the pathology report meets the highest standards.

A brief overview of nonneoplastic hepatic toxicity in fish.

Biochemical assays are not routinely used to assess liver damage in fish, therefore, a histopathological evaluation is usually required to determine the existence or extent of nonneoplastic liver toxicity. Many mammalian pathologists may be uncomfortable when requested to identify and interpret subtle liver changes in these unfamiliar animals. It may be reassuring to note that there are more similarities than differences between fish and mammals in terms of their macro- and microanatomy, physiological and biochemical characteristics, and pathologic responses to hepatotoxic substances. This brief overview addresses several topics pertaining to hepatotoxicity in fish, including: anatomic considerations, that is, how the anatomy of the fish liver may be predictive of its metabolic capacity, and also its microscopic appearance, following exposure to toxins; physiologic considerations, including comparisons between mammalian and fish livers regarding the uptake, elimination, toxification, or detoxification of xenobiotic compounds; morphologic responses to toxicity, in which some of the general types of findings that are most commonly observed in cases or studies of fish hepatotoxicity are highlighted; and last, responses of the fish liver to specific hepatotoxins.

Shark Cartilage, Cancer and the Growing Threat of Pseudoscience

The promotion of crude shark cartilage extracts as a cure for cancer has contributed to at least two significant negative outcomes: a dramatic decline in shark populations and a diversion of patients from effective cancer treatments. An alleged lack of cancer in sharks constitutes a key justification for its use. Herein, both malignant and benign neoplasms of sharks and their relatives are described, including previously unreported cases from the Registry of Tumors in Lower Animals, and two sharks with two cancers each. Additional justifications for using shark cartilage are illogical extensions of the finding of antiangiogenic and anti-invasive substances in cartilage. Scientific evidence to date supports neither the efficacy of crude cartilage extracts nor the ability of effective components to reach and eradicate cancer cells. The fact that people think shark cartilage consumption can cure cancer illustrates the serious potential impacts of pseudoscience. Although components of shark cartilage may work as a cancer retardant, crude extracts are ineffective. Efficiencies of technology (e.g., fish harvesting), the power of mass media to reach the lay public, and the susceptibility of the public to pseudoscience amplifies the negative impacts of shark cartilage use. To facilitate the use of reason as the basis of public and private decision-making, the evidence-based mechanisms of evaluation used daily by the scientific community should be added to the training of media and governmental professionals. Increased use of logical, collaborative discussion will be necessary to ensure a sustainable future for man and the biosphere.

Documented and potential research impacts of subclinical diseases in zebrafish.

The zebrafish (Danio rerio) has become a very important animal model in biomedical research. In contrast with other models, such as mice, there has been relatively little documentation or control of subclinical disease in zebrafish research facilities. Several infectious and noninfectious conditions are consistently detected by histopathology in apparently healthy D. rerio. The most commonly observed infectious agent in zebrafish is Pseudoloma neurophilia, which is a microsporidian organism that targets the central nervous system, peripheral nerves, and occasionally other tissues. Mycobacteriosis, caused by Mycobacterium chelonae and other species, is also a frequent finding. Less commonly encountered agents include Pseudocapillaria tomentosa, which can cause extensive proliferative enteritis, and a myxozoan (Myxidium sp.) that inhabits the urinary tract but appears to cause few if any pathological changes. Noninfectious diseases that are often clinically unapparent in zebrafish include hepatic megalocytosis, bile and pancreatic ductal proliferation, and neoplasms of the ultimobranchial gland, gastrointestinal tract, and testis. To date, there is little information on the degree to which these conditions may impact research in subclinically affected fish, but there is reason to believe that they should be considered as potentially significant causes of nonprotocol variation in experiments. Therefore, it is imperative that research facilities monitor their stocks for the presence of these occult diseases and be aware of their existence when interpreting study results. Furthermore, for underlying disease conditions that cannot be readily eradicated, it is essential to determine the physiological and immunological changes that they elicit in zebrafish. Understanding the cause, modes of transmission, and distribution of the pathogens would provide useful information for the development of control and prevention strategies.

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Safety of Aquaflor (Florfenicol, 50% Type A Medicated Article), Administered in Feed to Channel Catfish, Ictalurus punctatus

Aquaflor, a feed premix containing the broad spectrum antibacterial agent florfenicol (50% w/w), is being developed for use to control enteric septicemia (ESC) in channel catfish Ictalurus punctatus caused by the gram-negative enterobacterium Edwardsiella ictaluri. The recommended dose of Aquaflor to control ESC is 10 mg/kg body weight (BW)/day for 10 days. The study objective was to determine the safety of Aquaflor administered in feed to channel catfish at doses of 0 (control), 10, 30, and 50 mg/kg BW/day for 20 consecutive days. Parameters evaluated included daily mortality, behavioral (appetite, distribution, flight/fright response), and water chemistry observations, initial and terminal weight measurements, and gross and microscopic pathology. Medicated feed consumption was 67—86% of target with group mean doses of 8.5 mg/kg BW/day, 24.6 mg/kg BW/day, and 34.9 mg/kg BW/day. There were no mortalities or clinically observable changes noted at any of the dose levels tested. Aquaflor-related changes were limited to the food consumption and histopathology data. Although Aquaflor-related decreased feed consumption was noted in the 30 and 50 mg/kg BW/day groups, there were no differences in fish growth among the treatment groups. Aquaflor-related histopathology findings were limited to a histomorphologically evident dose-dependent decrease in hematopoietic/lymphopoietic tissue in the anterior kidneys, posterior kidneys, and spleens of channel catfish.

Intersex in Japanese Medaka (Oryzias latipes) Used As Negative Controls In Toxicologic Bioassays: A Review of 54 Cases From 41 Studies

Histologic assessment of the gonads to detect intersex has become a valuable end point in reproductive toxicologic testing for fish, and many studies have solidly linked intersex with exposure to endocrine active substances (EAS). An assumption in such studies is that spontaneous intersex does not occur in control fish. Using historical data derived from toxicologic tests with Japanese medaka (Oryzias latipes), we report a retrospective study in which we identified 54 individual instances of intersex (testicular oocytes or ovarian testicular tissue) in control medaka from 15 of 41 selected toxicologic studies. These studies, comprised of previously unpublished data, had been conducted at three geographically distant laboratories, each of which utilized unique water sources, employed somewhat different culture protocols, and maintained distinct medaka breeding colonies. During our histologic examinations, we also identified three germ cell neoplasms that had been inadvertently diagnosed as intersex. In the present report, we review potential causes of intersex, discuss possible reasons why spontaneous intersex has rarely been reported, and propose suggestions for the judicious interpretation of intersex results in medaka studies involving EAS.

Transforming growth factor-β response to mycobacterial infection in striped bass Morone saxatilis and hybrid tilapia Oreochromis spp.

Striped bass (Morone saxatilis) and hybrid tilapia (Oreochromis spp.) were experimentally infected with Mycobacterium marinum. Splenic mononuclear cell transforming growth factor-beta (TGF-beta) mRNA was measured by reverse transcription quantitative-competitive PCR (RT-qcPCR). In histologic sections of liver and anterior kidney, the area of each section that was occupied by granulomas and the total area of each section were measured by computer-assisted image analysis and compared as a proportion (the granuloma proportion). Infected striped bass splenic mononuclear cell TGF-beta mRNA expression was significantly lower than uninfected controls, while for tilapia there was no significant difference between infected and control fish. Mycobacterial granuloma proportion of liver and anterior kidney sections was significantly greater for infected striped bass than tilapia. Three (of 10) infected tilapia with the most pronounced inflammatory response displayed a decrease in TGF-beta mRNA expression, similar to the overall striped bass response to mycobacterium challenge. Downregulation of TGF-beta and failure to modulate the immune response may be related to excessive inflammatory damage to organs observed in mycobacteria-sensitive fish species.

Effects of 17 β-estradiol exposure on Xenopus laevis gonadal histopathology.

The natural estrogen 17 beta-estradiol (E2) is a potential environmental contaminant commonly employed as a positive control substance in bioassays involving estrogenic effects. The aquatic anuran Xenopus laevis is a frequent subject of reproductive endocrine disruptor research; however, histopathological investigations have tended to be less than comprehensive. Consequently, a study was designed to characterize gross and microscopic changes in the gonads of X. laevis as a result of E2 exposure. Additional goals of this study, which consisted of three separate experiments, included the standardization of diagnostic terminology and criteria, the validation of statistical methodology, and the establishment of a half maximal effective concentration (EC50) for E2 as defined by an approximately 50% conversion of presumptive genotypic males to phenotypic females. In the first experiment, frogs were exposed to nominal concentrations of 0, 0.2, 1.5, or 6.0 microg/L E2. From these experimental results and those of a subsequent range finding trial, the EC50 for E2 was determined to be approximately 0.2 microg/L. This E2 concentration was utilized in the other two experiments, which were performed at different facilities to confirm the reproducibility of results. Experiments were conducted according to Good Laboratory Practice guidelines, and the histopathologic evaluations were peer reviewed by an independent pathologist. Among the three trials, the histopathological findings that were strongly associated with E2-exposure (p<0.001 to 0.0001) included an increase in the proportion of phenotypic females, mixed sex, dilated testis tubules, dividing gonocytes in the testis, and dilated ovarian cavities in phenotypic ovaries. A comparison of the gross and microscopic evaluations suggested that some morphologic changes in the gonads may potentially be missed if studies rely entirely on macroscopic assessment.

Safety of Florfenicol Administered in Feed to Tilapia (Oreochromis sp.)

The safety of Aquaflor® (50% w/w florfenicol [FFC]) incorporated in feed then administered to tilapia for 20 days (2× the recommended duration) at 0, 15, 45, or 75 mg/kg body weight/day (0, 1, 3, or 5× the recommended dose of 15 mg FFC/kg BW/d) was investigated. Mortality, behavioral change, feed consumption, body size, and gross and microscopic lesions were determined. Estimated delivered doses were >96.9% of target. Three unscheduled mortalities occurred but were considered incidental since FFC-related findings were not identified. Feed consumption was only affected during the last 10 dosing days when the 45 and 75 mg/kg groups consumed only 62.5% and 55.3% of the feed offered, respectively. There were significant, dose-dependent reductions in body size in the FFC-dose groups relative to the controls. Treatment-related histopathological findings included increased severity of lamellar epithelial hyperplasia, increased incidence of lamellar adhesions, decreased incidence of lamellar telangiectasis in the gills, increased glycogen-type and lipid-type hepatocellular vacuolation in the liver, decreased lymphocytes, increased blast cells, and increased individual cell necrosis in the anterior kidney, and tubular epithelial degeneration and mineralization in the posterior kidney. These changes are likely to be of minimal clinical relevance, given the lack of mortality or morbidity observed. This study has shown that FFC, when administered in feed to tilapia at the recommended dose (15 mg FFC/kg BW/day) for 10 days would be well tolerated.