Effects of Chronic Ochratoxin A Exposure on p53 Heterozygousand p53 Homozygous Mice

GENEVIEVE S. BONDY1, DONALD S. CALDWELL

1, SYED A. AZIZ1, LAURIE C. COADY

1, CHERYL L. ARMSTRONG1,

IVAN H. A. CURRAN1, ROBYN L. KOFFMAN

2, KAMLA KAPAL1, DAVID E. LEFEBVRE

1, AND REKHA MEHTA1

1Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, ON, Canada2Regions and Programs Bureau, Health Canada, Burnaby, BC, Canada

ABSTRACT

Exposure to the mycotoxin ochratoxin A (OTA) causes nephropathy in domestic animals and rodents and renal tumors in rodents and

poultry. Humans are exposed to OTA by consuming foods made with contaminated cereal grains and other commodities. Management of

human health risks due to OTA exposure depends, in part, on establishing a mode of action (MOA) for OTA carcinogenesis. To further inves-

tigate OTA’s MOA, p53 heterozygous (p53þ/�) and p53 homozygous (p53þ/þ) mice were exposed to OTA in diet for 26 weeks. The former

are susceptible to tumorigenesis upon chronic exposure to genotoxic carcinogens. OTA-induced renal damage but no tumors were observed in

either strain, indicating that p53 heterozygosity conferred little additional sensitivity to OTA. Renal changes included dose-dependent

increases in cellular proliferation, apoptosis, karyomegaly, and tubular degeneration in proximal tubules, which were consistent with ochra-

toxicosis. The lowest observed effect level for renal changes in p53þ/� and p53þ/þ mice was 200 mg OTA/kg bw/day. Based on the lack of

tumors and the severity of renal and body weight changes at a maximum tolerated dose, the results were interpreted as suggestive of a pri-

marily nongenotoxic (epigenetic) MOA for OTA carcinogenesis in this mouse model.

Keywords: ochratoxin; p53; mouse; carcinogenicity; chronic toxicity.

INTRODUCTION

Ochratoxin A (OTA) was first identified and structurally

characterized from a culture of Aspergillus ochraceus Wilh.

isolated from sorghum (van der Merwe et al. 1965) and is now

known to be produced by species in the fungal genera Aspergil-

lus and Penicillium (Varga et al. 2001). It is a phenylalanyl

derivative of a substituted isocoumarin (R)-N-[5-chloro-3,4-

dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-y1)-

carbonyl]-L-phenylalanine (Marin et al. 2013, van der Merwe

et al. 1965). Although ochratoxigenic fungi may be present

on crops at harvest, OTA is primarily produced in storage when

crop moisture content, temperature, and other environmental

conditions favor fungal growth (Ominski et al. 1994). OTA is

widely distributed in food commodities including cereal grains,

dried fruits, and spices, coffee, beer, wine, cocoa, and meats

(Marin et al. 2013). Relative to other foods, consumption of

OTA in cereal grain–based foods contributes significantly to

human dietary OTA exposure (Duarte, Pena, and Lino 2010;

Kuiper-Goodman et al. 2010).

Exposure to OTA is nephrotoxic in rodents, pigs, and poul-

try, and causes renal tumors in rodents and poultry (Battacone,

Nudda, and Pulina 2010; Bendele et al. 1985; Boorman et al.

1992; Krogh et al. 1979; Stoev 2010). Thus, OTA has long

been a suspected contributor to human nephropathies. Expo-

sure to OTA in food has been linked to Balkan endemic

nephropathy (BEN), a chronic progressive renal disease associ-

ated with upper urothelial tract tumors that occur in rural popu-

lations along the Danube River (Pfohl-Leszkowicz 2009). In

this case, OTA involvement is controversial and there is also

evidence supporting the carcinogenic plant toxin aristolochic

acid as a causative agent of BEN (Grollman et al. 2007; Stefa-

novic, Polenakovic, and Toncheva 2011; Wu and Wang 2013).

A recent review of studies linking OTA to human nephropa-

thies concluded that there was no statistically significant evi-

dence for human health risks associated with OTA exposure.

However, the analysis was based on three studies that met the

established criteria for review, underscoring the need identified

by the authors for larger cohort or case-control OTA

The author(s) declared no potential conflicts of interest with respect to the

research, authorship, and/or publication of this article.

The author(s) received no financial support for the research, authorship,

and/or publication of this article.

Address correspondence to: Genevieve Bondy, Toxicology Research

Division 2202D, Bureau of Chemical Safety, Food Directorate, Health

Products and Food Branch, Health Canada, Ottawa, ON, Canada K1A 0K9;

e-mail: genevieve.bondy@hc-sc.gc.ca.

Abbreviations: AFIP, Armed Forces Institute of Pathology; ANOVA,

analysis of variance; BEN, Balkan endemic nephropathy; BW, body weight;

CLU, clusterin; D-PBS, Dulbecco’s phosphate buffered saline; DSB, double

strand break; ECP, eosinophilic crystalline pneumonia; EDTA,

ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbant

assays; EFSA, European Food Safety Authority; HR, homologous

recombination; 8-OHdG, 8-hydroxy-20-deoxyguanosine; IARC, International

Agency for Research on Cancer; Ig, immunoglobulin; JECFA, Expert

Committee on Food Additives; LOEL, lowest observed effect level; MOA,

mode of action; NIEHS, National Institute of Environmental Health

Sciences; NTP, National Toxicology Program; OPN, osteopontin; OSOM,

outer stripe of the outer medulla; OTA, ochratoxin A; p53þ/�, p53

heterozygous; p53þ/þ, p53 homozygous; PBS, phosphate buffered saline

solution; PCNA, proliferating cell nuclear antigen; PE-Cy5.5, phycoerythrin-

cyanine 5.5; PWM, pokeweed mitogen; TUNEL, terminal deoxynucleotidyl

transferase dUTP nick end labeling; WHO, World Health Organization.

1

Toxicologic Pathology, XX: 1-15, 2015

Copyright # 2015 by The Author(s)

ISSN: 0192-6233 print / 1533-1601 online

DOI: 10.1177/0192623314568391

epidemiology studies and for studies validating biomarkers of

human OTA exposure (Bui-Klimke and Wu 2014). Taken

together, the classification of OTA by the International Agency

for Research on Cancer (IARC) as a possible human carcino-

gen with sufficient evidence for carcinogenicity in experimen-

tal animals but inadequate evidence in humans (IARC 1993) is

consistent with current knowledge. Although nephropathy is

the primary noncancer outcome in OTA-exposed animals,

establishing a link between chronic kidney disease and OTA

exposure in humans is complicated by the multifactorial nature

of renal disease (Jha et al. 2013; McClellan and Flanders 2003)

and the lack of knowledge on OTA’s exact MOA.

Multiple MOAs for OTA-induced renal tumor formation

and nephropathy have been proposed based on in vivo and in

vitro studies. Although OTA exposure leads to DNA damage,

there is controversy about whether this occurs due to direct

interaction with DNA by OTA or a reactive OTA metabolite

(genotoxicity), or indirectly due to interaction of OTA or a

metabolite with non-DNA targets (nongenotoxicity). A cova-

lent DNA adduct (C-C8 OTA 30 dGMP) with mutagenic poten-

tial has been detected in kidneys from rats exposed to OTA,

although independent laboratories have not confirmed these

findings (Delatour et al. 2008; Mantle et al. 2010; Sharma,

Manderville, and Wetmore 2013). In support of an indirect

MOA, in vivo and in vitro studies indicate that OTA exposure

increases the production of reactive oxygen species (ROS)

leading to oxidative DNA damage in renal cells (Marin-Kuan

et al. 2011). There is also evidence that OTA disrupts antioxi-

dant defense responses regulated by the transcription factor

nuclear factor-erythroid 2-related factor 2 (Marin-Kuan et al.

2006), which could exacerbate ROS-induced oxidative stress

in kidney proximal tubule cells. Inhibition of gap junction

intercellular communication has been associated with cancer

development and has been shown to be disrupted by OTA in

vitro (Mally et al. 2006). Cell-based assays and transcriptomic

analyses of renal tissues and cultured cells indicate that OTA

can perturb posttranslational protein modifications that regu-

late many cellular functions (Jennings et al. 2012). These

include histone acetyltransferase inhibition leading to mitotic

disruption that may explain, in part, OTA-associated karyome-

galy, genetic instability, and tumorigenesis (Czakai et al. 2011;

Mally 2012).

Uncertainty in the MOA for OTA-induced DNA damage

impacts its risk assessment and management. Approaches for

food-borne carcinogens are based, in part, on the assumption

that there is no threshold for the dose response to genotoxic car-

cinogens, whereas homeostatic mechanisms impart a threshold

to the dose response elicited by nongenotoxic carcinogens

(Barlow et al. 2006). The Joint FAO/World Health Organiza-

tion (WHO) Expert Committee on Food Additives (JECFA)

considered that the weight of scientific evidence pointed

to multiple nongenotoxic MOAs for OTA (JECFA 2008).

Kuiper-Goodman et al. (2010) considered the genotoxicity of

OTA to be equivocal and based their health risk assessment

on genotoxicity, which is a default position for a substance with

an unknown carcinogenic MOA and not an acknowledgment

that genotoxicity per se is the MOA (European Food Safety

Authority [EFSA] 2005). While many in vitro and in vivo stud-

ies contribute to determining OTA’s MOA, multidose chronic

exposure and carcinogenesis studies provide the most valuable

dose–response data for risk assessment. Two-year carcinogen-

esis studies using F344/N and B6C3F1 mice are key studies for

OTA risk assessment (Bendele et al. 1985; JECFA 2008;

Kuiper-Goodman et al. 2010; NTP 1989). The present 26-

week study, a comparison of heterozygous p53þ/� mice and

corresponding p53þ/þ mice exposed to OTA in diet, provides

further data on the chronic toxicity and carcinogenicity of OTA

over a dose range relevant for risk assessment.

Transgenic mice heterozygous for a null p53 allele display

increased susceptibility to a wide range of carcinogens and a

low spontaneous tumor incidence rate up to about 9 months

in age (French, Storer, and Donehower 2001b). They have been

used as a model to identify potential chemical carcinogens

(Donehower et al. 1992; French et al. 2001a). The p53 protein

acts as a transcription factor that regulates multiple genes

involved in cellular functions and genome maintenance (Harris

and Levine 2005). The U.S. National Institute of Environmen-

tal Health Sciences/National Toxicology Program (NTP) eval-

uated the p53þ/� mouse for use in short-term carcinogenicity

testing and observed decreased latency and increased suscept-

ibility to tumor formation by genotoxic carcinogens (Dunnick

et al. 1997; French et al. 2001a; French, Storer, and Donehower

2001b; Pritchard et al. 2003; Tennant, Spalding, and French

1996). Previous chronic dietary exposure studies using the

p53þ/� mouse in our laboratory supported a nongenotoxic

MOA for the carcinogenic fungal toxin fumonisin B1, which

was consistent with the disruption of sphingolipid metabolism

and cellular regulators of apoptosis and proliferation rather

than direct genotoxicity (Bondy et al. 2012). In this study, the

p53þ/� mouse model was used to provide further insight into

the MOA for OTA carcinogenesis.

METHODS AND MATERIALS

Toxin and Diet Preparation

OTA (>98% purity; benzene free) was purchased from

Sigma-Aldrich Canada (Oakville, ON, Canada). All mice

received a modified AIN-93M diet (Dyets Inc., Bethlehem,

PA) during the acclimation period and throughout the study.

To prepare test diets, OTA was dissolved in methanol and

added to the diet. The resulting slurry was mixed thoroughly

while the methanol was removed by evaporation to dryness,

resulting in a stock diet containing 1.6 g of OTA/kg diet. The

OTA stock diet was mixed with control diet using an industrial

food mixer to prepare test diets containing 1-, 15-, or 40-mg

OTA/kg diet (weeks 1 and 2) and 0.5-, 2-, or 10-mg OTA/kg

diet (weeks 3–26).

Diet samples were collected monthly during the study to

verify OTA levels in treatment diets and to detect background

OTA levels in the control diet. Feed analyses methods were

adapted from Lombaert et al. (2003). Briefly, feed samples

were extracted with acetonitrile, methanol, and water (1:1:2)

2 BONDY ET AL. TOXICOLOGIC PATHOLOGY

by volume; centrifuged; and filtered. Aliquots of each sample

were diluted with phosphate buffered saline solution (PBS) and

cleaned up using an LCTech OTAClean immunoaffinity col-

umn (Dorfen, Germany) prior to OTA determination by liquid

chromatography (LC) with fluorescence detection. A 50-ml

volume was injected onto the LC system consisting of a Waters

Alliance 2695 liquid chromatograph (Milford, MA) combined

with Waters 2475 multiwavelength fluorescence detector and a

150 mm� 2.1 mm (3 mm) reversed phase Waters Atlantis dc18

analytical column. The fluorescence detector was set to 333 nm

excitation and 460 nm emission wavelengths.

The OTA primary reference standard (Sigma-Aldrich

Canada, Oakville, Ontario, Canada) was prepared in ben-

zene:acetic acid (99:1). Calibration standard solutions were

prepared in acetonitrile/water/acetic acid (99:99:2) by volume

ranging from 1 to 20 ng/ml, representing 2 to 40 ng/g in sam-

ples. The limit of quantitation for OTA was 6 ng/g. This was

determined as that level of standard in sample matrix producing

a chromatographic signal/noiseptp ratio of 10. The limit of

detection for OTA was 1.6 ng/g, determined with a chromato-

graphic signal/noiseptp ratio of 3.

Recovery experiments were performed with each sample set

using a known negative rodent feed sample. Samples were

spiked at 4 ng/g. The mean recovery for OTA was 108% (SD

¼ 2.5%, n¼ 3). Qualitative confirmation of the analyte identity

was performed by methyl esterification. Following sample

analysis, an aliquot of each calibration standard and sample

solution was removed and methyl esterified. Methyl esterifica-

tion resulted in the diminution of the OTA chromatographic

peak and the appearance of a new chromatographic peak corre-

sponding to methyl-OTA.

Animals and Study Design

Forty male p53þ/� mice (P53N5-T) mice and 40 male

p53þ/þ (P53N5-W) mice, 5 to 7 weeks old, were purchased

from Taconic Farms Inc. (Germantown, NY). Mice were

weighed and individually caged upon arrival. Cages were

housed in semirigid HEPA-filtered isolator units manufactured

for Health Canada by Charles River (Boston, MA). Mice

received modified AIN-93M diet and water ad libitum for 1

week prior to the study while acclimating to the animal facility

and isolator units. Temperature and humidity inside the isolator

units were monitored daily for the duration of the study.

Throughout the study, mice received care and handling accord-

ing to the requirements of the Canadian Council for Animal

Care.

The mean body weight (BW) of all mice (p53þ/þ and

p53þ/�) upon arrival was 26.0 + 2.4 g. During acclimation,

p53þ/þ and p53þ/� mice were divided randomly into 4

groups of 10 mice/dose/strain. There were no significant differ-

ences in starting BWs across dose groups and strain at the

beginning of the study (Table 1). During the study, mice

received water and control or test diet ad libitum. BW and food

consumption data were collected weekly. At the beginning of

the study, p53þ/þ and p53þ/� mice received feed containing

0-, 1-, 15-, or 40-mg OTA/kg diet, as in Bendele et al. (1985).

By the end of week 2, BWs in p53þ/þ mice were 20% and

28% lower in the 15- and 40-mg/kg dose groups, respectively;

in p53þ/�mice, BWs were 27% and 31% lower in the 15- and

40-mg/kg dose groups, respectively (Figure 1A and B). Due to

rapid weight loss, test diets were removed from the p53þ/þand p53þ/�medium- and high-dose groups at the end of week

2 and replaced with control diet for 24 hr. After 24 hr, reformu-

lated diets were given to all test groups (Figure 1). For the

remainder of the study, mice received 0-, 0.5-, 2-, or 10-mg

OTA/kg diet. After 26 weeks, final BWs were recorded and all

mice were exsanguinated by cardiac puncture under isoflurane

anesthesia (Baxter Corporation, Mississauga, ON, Canada).

Organ weights were recorded at necropsy for liver, kidney, thy-

mus, and spleen.

Hematology and Immunology

Whole blood was collected in ethylenediaminetetraacetic

acid (EDTA) at necropsy. White blood cells (WBCs), neutro-

phils, lymphocytes, monocytes, eosinophils, and basophils were

enumerated using a Coulter AcTTM 5 Diff Cap Pierce Hematol-

ogy Analyzer (Beckman Coulter, Mississauga, ON, Canada).

Immunophenotype analysis of whole blood by flow cyto-

metry was based on four color panels using the following

monoclonal antibodies (BD Biosciences, Mississauga, ON,

TABLE 1.—Initial BW, final BW, and final organ weights of p53þ/þ and p53þ/� mice exposed to dietary OTA for 26 weeks.

p53þ/þ p53þ/�

OTA added to diet

(mg OTA /kg diet)a 0 0.5 2 10 0 0.5 2 10

Initial BW (g) 28.8 + 3.9 28.4 + 3.0 30.2 + 3.3 30.3 + 2.6 28.7 + 3.1 29.3 + 2.5 28.4 + 1.7 28.2 + 1.8

Final BW (g) 48.0 + 4.9 46.5 + 3.3 42.4 + 4.4b 33.5 + 3.9b 45.7 + 3.5 46.8 + 3.1 39.4 + 2.7b 28.6 + 2.9b

Liver (% BW) 5.95 + 0.85 5.37 + 0.48 4.59 + 0.51b 4.37 + 0.34b 5.52 + 0.84 5.47 + 0.43 4.46 + 0.42b 4.27 + 0.24b

Kidney (% BW) 0.43 + 0.04 0.42 + 0.03 0.40 + 0.05 0.35 + 0.02b 0.41 + 0.02 0.40 + 0.02 0.40 + 0.02 0.37 + 0.02b

Spleen (% BW) 0.23 + 0.02 0.23 + 0.05 0.23 + 0.02 0.28 + 0.04b 0.20 + 0.02 0.21 + 0.03 0.21 + 0.01 0.27 + 0.03b

Thymus (% BW) 0.09 + 0.02 0.08 + 0.02 0.08 + 0.01 0.10 + 0.01 0.08 + 0.01 0.09 + 0.02 0.09 + 0.02 0.11 + 0.02b

Note: OTA ¼ ochratoxin A; BW ¼ body weight.aDietary OTA levels for week 3 to 26. bData are significantly different from corresponding controls using analysis of variance (ANOVA) followed by post hoc pairwise comparisons

using the Holm–Sidak or Dunn’s test (p � .05).

Vol. XX, No. X, 2015 OCHRATOXIN A EFFECTS ON P53þ/� MICE 3

Canada): rat anti-mouse CD45 phycoerythrin-cyanine 5.5 (PE-

Cy5.5) clone 30-F11, Leucocyte Common Antigen; hamster

anti-mouse CD3e fluorescein isothiocyanate clone 145-2C11,

T lymphocyte; rat anti-mouse CD4 allophycocyanine clone

RM4-5, T helper lymphocyte; rat anti-mouse CD8a R-

phycoerytherin clone 53-6.7, T cytotoxic lymphocyte; rat

anti-mouse CD19 allophycocyanine clone 1D3, B lymphocyte;

rat anti-mouse CD49b phycoerythrin clone DX5, natural killer

cell. Blood samples were mixed with antibodies and incubated

for 10 min protected from light at room temperature. Red blood

cells were lysed using a TQ-Prep (Beckman Coulter, Missis-

sauga, ON, Canada) and Immunoprep lysis reagent (Beckman

Coulter, Brea, CA). Lymphocyte subset analyses were per-

formed by flow cytometry using a Becton Dickinson FACSCa-

libur System equipped with 488 nm and 635 nm air-cooled

lasers (BD Biosciences, Mississauga, ON, Canada).

Splenocyte suspensions for immunophenotyping were pre-

pared in Dulbecco’s phosphate buffered saline (D-PBS;

Gibco/Invitrogen, Grand Island, NY) by gently crushing tissues

in a 70-mm nylon cell strainer (BD Falcon, Bedford, MA) and

rinsing into a 50-ml tube. Prior to staining, WBCs were counted

using a Beckman Coulter AcTTM 5 Diff Cap Pierce Hematology

Analyzer and cell suspensions were standardized to 2 � 107

WBCs/ml. Cell staining was performed by mixing a 100 mL

aliquot of suspended splenocytes with antibodies as described

previously and fixing using 0.1% buffered formaldehyde.

Splenocytes for proliferation assays were released from the

spleen by gentle dispersion into D-PBS using tissue forceps.

Erythrocytes were removed by lysis using ammonium-

chloride-potassium lysing buffer (Quality Biological Inc.,

Gaithersburg, MD), splenocytes were strained with trypan

blue, and viable cells were enumerated using a hemocytometer.

Cultures containing 5 � 105 viable splenocytes in Gibco serum-

free AIM-V medium supplemented with 2-mercaptoethanol

(Invitrogen Canada Inc., Burlington, ON, Canada) were incu-

bated in the presence of pokeweed mitogen (Sigma) or medium

only (unstimulated controls) for 72 hr. Splenocyte proliferation

was measured by quantifying 6-hr 3H-methylthymidine

(1 mCi/ well) incorporation using a LKB Wallac liquid scintil-

lation counter (Perkin Elmer Life Sciences, Woodbridge, ON,

Canada).

Total plasma immunoglobulin levels were analyzed by

sandwich enzyme-linked immunosorbant assays (ELISA)

using primary and secondary antibodies specific for mouse

IgA, IgG, and IgM (Bethyl Laboratories Inc., Montgomery,

TX). Sandwich ELISAs were performed according to the pro-

tocol accompanying the antibodies using buffers and reagents

as described by Tryphonas et al. (2004).

Histology

A gross visual examination was performed on each mouse at

necropsy. Liver, right kidney, thymus, spleen, esophagus, tra-

chea, thyroid, stomach (including forestomach), ileum (includ-

ing Peyer’s patches), mesenteric lymph nodes, popliteal lymph

nodes, heart, and lungs were fixed by immersion in 10% neutral

buffered formalin. Samples from the fixed tissues were paraffin

embedded and blocked. The tissue blocks were sectioned to 5

mm, stained with hematoxylin and eosin according to the

method described by Luna (1968), and examined by light micro-

scopy. Renal lesions were classified according to International

Harmonization of Nomenclature and Diagnostic Criteria nomen-

clature (Frazier et al. 2012). Lesion severity was graded subjec-

tively in comparison to control animals and assigned a numeric

score from 0 to 5 where 0 indicated that a lesion was not present

or present within subjective normal limits, and where 1 through

5 indicated that lesions were minimal, mild, moderate, marked,

and severe, respectively. Lymphoid tissues were assessed for

direct immunotoxicity according to International Collaborative

Immunotoxicity Study (1998). Testes were fixed by immersion

in Bouin’s fixative at necropsy. After 24 hr, they were washed

and transferred to 70% ethanol. Fixed testes were trimmed to

produce transverse sections at the level of rete and then

embedded in paraffin. Tissue blocks were sectioned to 5 mm and

stained with periodic acid Schiff hematoxylin. A section from

the right testes of each mouse was evaluated by light microscopy

for standard lesions (Foley 2001; Lanning et al. 2002).

Renal Immunohistochemistry

Unstained kidney sections (5 mm thick) were used for immu-

nohistochemical procedures. To detect proliferating cells, sec-

tions were first subjected to antigen retrieval by heating in 10

mmol/L sodium citrate buffer in a microwave for 2.5 min. The

mouse monoclonal anti-proliferating cell nuclear antigen

(PCNA) antibody (DakoCytomation, Carpinteria, CA), diluted

1:10,000, was used as the primary antibody. The EnVisionTM

þ System (DakoCytomation), using an anti-mouse secondary

antibody conjugated with horseradish peroxidase followed by

detection with diaminobenzidine, was applied according to the

manufacturer’s instructions to visualize PCNA-positive cells.

Harris’s hematoxylin was used as the counterstain.

For apoptosis, the ApopTag1 Plus Peroxidase In Situ Apop-

tosis Kit (Chemicon, Temecula, CA) was used according to the

manufacturer’s instructions. This terminal deoxynucleotidyl

transferase dUTP nick end labeling (TUNEL) assay-based

method detects early apoptosis via DNA fragmentation by enzy-

matically labeling the free 30-OH termini with modified nucleo-

tides. Briefly, sections were subjected to a protein digestion

enzyme to quench endogenous peroxidase, followed by applica-

tion of an equilibration buffer and incubation with TdT enzyme,

before stopping the reaction by adding anti-digoxigenin

FIGURE 1.—Time line for exposure of p53þ/þ and p53þ/� mice to

ochratoxin A in diet.

4 BONDY ET AL. TOXICOLOGIC PATHOLOGY

conjugate buffer. This was followed by incubation with the per-

oxidase substrate and counterstain with methyl green.

For clusterin (CLU) detection, sections were first subjected

to antigen retrieval by heating in Tris EDTA, pH 9, in a 100�Cwater bath for 30 min. Sections were incubated with the pri-

mary antibody rabbit polyclonal anti-CLU (Santa Cruz Bio-

technology, Dallas, TX) diluted 1:100, followed by detection

with the EnVisionTM þ System (DakoCytomation) and Har-

ris’s hematoxylin counterstain.

To detect osteopontin (OPN), kidney sections were first sub-

jected to antigen retrieval by heating in a commercially

obtained antigen retrieval solution (S1700; Dako Cytomation)

in a water bath at 100�C for 30 min. After blocking endogenous

biotin, biotin receptor, and avidin binding sites (Avidin/biotin

blocking kit, Vector Laboratories, Burlingame, CA), sections

were incubated with the primary antibody rabbit polyclonal

anti-OPN (Abcam Inc., Cambridge, MA) diluted 1:250, fol-

lowed by detection with the EnVisionTM þ System (Dako

Cytomation) and Harris’s hematoxylin as the counterstain.

The immunostained sections were analyzed using a Zeiss

Axiophot light microscope (Carl Zeiss Canada Ltd., Toronto,

ON, Canada) and image analysis. Proliferating (PCNA-posi-

tive) and apoptotic (TUNEL-positive) cells were enumerated

in proximal tubules for 1 entire kidney section per mouse. The

ratios of proliferating cells (PCNA-positive) or apoptotic cells

(TUNEL-positive) per section area (mm2) were determined

using Northern Eclipse Version 7.0 software (Empix Imaging,

Inc., Mississauga, ON, Canada). Semiquantitative assessments

were conducted on CLU- and OPN-stained sections. Ten fields

per section covering most of the section area were assessed for

percentage proximal tubule staining in each field using North-

ern Eclipse version 7.0 software, generating a mean percentage

positivity score for 1 kidney section per mouse.

Statistical Analyses

Statistical comparisons were conducted using SigmaPlot 12

(Systat Software, Inc., San Jose, CA). Data were analyzed for nor-

mality using the Kolmogorov–Smirnov test. Equal variance was

tested by checking the variability around group means. Within

each strain (p53þ/þ or p53þ/�), multiple group comparisons

were conducted by 1-way analysis of variance (1-way ANOVA),

followed by post hoc pairwise comparisons using the Holm–

Sidak test. Multiple group comparisons of nonparametric data

were conducted using the Kruskal–Wallis ANOVA on Ranks,

followed by post hoc pairwise comparisons using the Dunn’s

method. Animal weights and relative food consumption (food

consumption/BW) were analyzed by ANOVA on weekly average

weights (Hoffman, Ness, and van Lier 2002). For histopathology

endpoints, including CLU and OPN data, lesion severity data

were analyzed using the Kruskal–Wallis 1-way ANOVA on

Ranks within each strain. Comparisons of data from p53þ/þ and

p53þ/� mice were conducted by 2-way ANOVA to identify

interactions between strain and dose, followed by post hoc pair-

wise comparisons using the Holm–Sidak method.

RESULTS

Diet Analyses

OTA was not detected in control diets. Levels of OTA

detected in treatment diets are summarized in Tables 2 and 3.

After correcting for recovery, OTA was recovered from treat-

ment diets at levels 20–30% lower than OTA added to diets

at preparation.

Effects of OTA on BW, Food Consumption, and Organ

Weight

In the first 2 weeks on diet, BW gain in all mice in the medium

(15-mg OTA/kg diet) and high (40-mg OTA/kg diet) dose

groups was significantly depressed compared to mice in the con-

trol and low (1 mg/kg) dose groups. By the end of week 2, BWs

in p53þ/þmice were 20% and 28% lower in the 15- and 40-mg/

kg dose groups, respectively; in p53þ/� mice, BWs were 27%and 31% lower in the 15- and 40-mg/kg dose groups, respec-

tively (Figure 2A and B). It was decided that medium- and

high-dose group mice would not survive the 26-week exposure

period, so reformulated diets containing lower OTA levels were

introduced at the beginning of week 3. From this time onward,

mice in the new medium (2-mg OTA/kg diet) and high (10 mg

OTA/kg diet) dose groups gained weight. Control p53þ/þ and

p53þ/� mice and mice in the new low-dose group (0.5-mg

OTA/kg diet) gained weight throughout the exposure period.

In both p53þ/þ and p53þ/�mice, BWs of low-dose mice were

not significantly different from respective controls at any time

(Figure 2A and B). Mean BWs of medium-dose p53þ/þ mice

were significantly lower than respective controls in week 2 and

weeks 6–26. Mean BWs of high-dose p53þ/þ mice were

TABLE 2.—Ochratoxin A consumption by p53þ/þ and p53þ/� mice in week 1 of the 26-week exposure period.

p53þ/þ p53þ/�

OTA added to diet (mg OTA /kg diet) 0 1 15 40 0 1 15 40

OTA analyzed in diet (mg OTA/kg diet)a <4 � 10�4 0.8 10.2 31.7 <4 � 10�4 0.8 10.2 31.7

Mean daily OTA consumption based on

OTA recovered from diet (mg OTA/kg

BW/day)

0 0.12 + 0.02 1.44 + 0.49 4.42 + 1.52 0 0.09 + 0.01 1.30 + 0.23 3.94 + 1.21

Note: OTA ¼ ochratoxin A; BW ¼ body weight.aOne feed sample from week 1 for each diet/dose group was analyzed as described in the Methods and Materials section.

Vol. XX, No. X, 2015 OCHRATOXIN A EFFECTS ON P53þ/� MICE 5

significantly lower than respective controls from weeks 1 to 26

(Figure 2A). In p53þ/� mice, mean BWs of medium and high

dose mice were significantly lower than respective controls

from weeks 1 to 26 (Figure 2B).

The effect of OTA exposure on BW-normalized food con-

sumption (relative food consumption) was evaluated for each

week of the study (Figure 3A and B). In p53þ/þ medium-

dose mice, relative food consumption was significantly higher

than in respective controls from weeks 6 to 11; in p53þ/þhigh-dose mice, relative food consumption was significantly

higher than in respective controls in week 3 and weeks 4 to

26 (Figure 3A). In p53þ/� medium-dose mice, relative food

consumption was higher than in respective controls in weeks

3, 4, 6, 10, 11, 14 to 17, 19, and 21 to 23. In p53þ/� high-

dose mice, relative food consumption was significantly higher

than in respective controls from weeks 1 to 26 (Figure 3B).

Mean daily OTA consumption values were calculated based

on OTA levels detected in diets, food consumption, and BW

(Tables 2 and 3). In the 1st week of the study, mean daily OTA

consumption values were 0.12-, 1.44-, and 4.42-mg OTA/kg

bw/day for p53þ/þ low-, medium-, and high-dose diets,

respectively. For p53þ/� low-, medium- and high-dose

groups, mean daily OTA consumption values were 0.09-,

1.30-, and 3.94-mg OTA/kg bw/day, respectively. Calculations

were not possible for week 2 due to feed refusal and feed

wastage in the medium- and high-dose groups (Table 2); how-

ever, similar calculations were done for the remaining 24

weeks of the study (Table 3). Mean daily OTA consumption

increased over week 3 to 24, in parallel with increasing relative

food consumption. This resulted in a range of mean daily OTA

consumption values calculated for weeks 3 to 26 which were

lowest early in the study and gradually increased to the highest

value by the end of the study (Table 3).

Final organ weights expressed as percentage BW are sum-

marized in Table 1. Liver weights were significantly reduced

relative to respective controls in p53þ/þ and p53þ/� mice

in the medium- and high-dose groups, and kidney weights were

significantly reduced in p53þ/þ and p53þ/�mice at the high-

est dose. Spleen weights were significantly increased in p53þ/

þ and p53þ/� mice in the high-dose group. Relative thymus

weights were significantly higher relative to controls in

p53þ/� mice in the high-dose group.

Effects of OTA on Immune Parameters

There were no significant changes due to OTA in numbers

of circulating WBCs, lymphocytes, lymphocyte subsets, or

granulocytes (data not shown). There were no significant

changes in splenocyte subpopulations due to OTA exposure

(data not shown). Splenocyte proliferation was significantly

reduced in p53þ/þ mice in the high-dose group relative to

controls (Figure 4). Plasma total immunoglobulin M, G, and

A levels were unaffected by OTA in p53þ/þ and p53þ/�mice

(data not shown).

Histopathologic Changes in Mice Exposed to OTA

Hepatocytes were markedly enlarged with cytoplasmic

clearing and vacuolation in the livers of control and low-dose

mice of both strains, which was generally associated with

relatively high body fat levels. In medium-dose p53þ/þ and

p53þ/� mice, hepatocytes were less swollen with less

cytoplasmic clearing and vacuolation. In high-dose mice,

hepatocyte cytoplasmic swelling with vacuolation was not

observed. Occasional foci of hepatocellular necrosis were

observed in all control and low-dose mice. The incidence and

severity of focal necrosis increased in medium- and high-

dose mice relative to controls. There were no strain-related dif-

ferences in the incidence and severity of hepatocellular necro-

sis (data not shown).

The incidence and severity of renal lesions in p53þ/þ and

p53þ/� mice are summarized in Table 4 and examples of typ-

ical renal lesions are shown in Figure 5. In both strains, micro-

scopic lesions were evident in renal tissues at all doses,

dominated by increasingly severe and prevalent karyomegaly,

apoptosis, and anisokaryosis affecting the proximal tubular

epithelium, including the S1 and S2 convoluted segments (cor-

tical labyrinth) and the S3 straight segments (outer stripe of the

outer medulla [OSOM] and medullary ray). Rare epithelial

cells with mild degeneration were present in the tubular epithe-

lium in the 0.5-mg OTA/kg dose group in both mouse strains.

TABLE 3.—Ochratoxin A consumption by p53þ/þ and p53þ/� mice from week 3 to 26 of a 26-week exposure period.

p53þ/þ p53þ/�

OTA added to diet (mg OTA /kg diet) 0 0.5 2 10 0 0.5 2 10

OTA analyzed in diet (mg OTA/kg diet)a <4 � 10�4 0.39 + 0.05 1.47 + 0.23 7.81 + 0.39 <4 � 10�4 0.39 + 0.05 1.47 + 0.23 7.81 + 0.39

Mean daily OTA consumption values for week 3

to 26 based on OTA recovered from diet (mg/

kg bw/day)b

0 0.03 + 0.01 0.20 + 0.06 1.46 + 0.35 0 0.03 + 0.00 0.19 + 0.05 1.55 + 0.42

Range of calculated mean daily OTA

consumption values for week 3 to 26 (mg/kg

BW/day)c

0 0.04 – 0.07 0.18 – 0.44 1.50 – 2.41 0 0.04 – 0.05 0.20 – 0.45 1.61 – 3.01

Note: OTA ¼ ochratoxin A; BW ¼ body weight.aMonthly feed samples from each dose group, n ¼ 6 samples total/dose group, were analyzed as described in the Methods and Materials section. OTA was not detected in control

diet. bReliable food consumption values for week 2 were unavailable due to feed refusal and coincident feed wastage. cOTA consumption gradually increased over time as mice gained

weight and consumed correspondingly more diet.

6 BONDY ET AL. TOXICOLOGIC PATHOLOGY

Mild tubular degeneration, evident as cytoplasmic vacuolation

with or without pale basophilic coloration, was observed in the

2-mg OTA/kg dose group in both mouse strains. In the 10-mg

OTA/kg dose groups, occasional regenerative tubules were

observed in the cortex, with mild tubular degeneration affect-

ing cortical tubular epithelium. No adenomas, carcinomas, or

cystic tubules lined by hyperplastic epithelium, as described

by Bendele et al. (1985), were detected.

There were no lesions in immune tissues, including thymus,

Peyer’s patches, spleen, and mesenteric and inguinal lymph

nodes. Mild degenerative lesions were observed in testicular

seminiferous tubules from all mice. These changes appeared

to be more prevalent in p53þ/þ than in p53þ/� mice across

all doses, although this observation was not quantified.

There were no lesions in any other tissues examined, with

the exception of the lungs. In the lungs of p53þ/þ and

p53þ/� mice in all dose groups, including controls, lesions

consistent with eosinophilic crystalline pneumonia (ECP) were

observed (Hoenerhoff, Starost, and Ward 2006). In control,

low-, and medium-dose mice, these lesions were characterized

by mild to marked lymphoid/ plasma cell infiltration in peri-

bronchiolar and perivascular regions. Scattered in the alveoli

were few macrophages containing abundant densely eosinophi-

lic cytoplasm that had a crystalline appearance. Large eosino-

philic rod-shaped crystals occurred occasionally in bronchiolar

lumens. In high-dose mice, similar lesions appeared to be more

FIGURE 2.—Mean body weights (BWs; g; þSD) in (A) p53þ/þ and

(B) p53þ/� mice exposed to ochratoxin A in diet for 26 weeks. Mean

BWs of medium-dose p53þ/þ mice were significantly lower than

respective controls in week 2 and weeks 6 to 26; mean BWs of

high-dose p53þ/þ mice were significantly lower than respective con-

trols from weeks 1 to 26 (A). Mean BWs of medium-and high-dose

p53þ/� mice were significantly lower than respective controls from

weeks 1–26 (B).

FIGURE 3.—Mean weekly feed consumption (g; þSD) in (A) p53þ/þand (B) p53þ/�mice exposed to ochratoxin A in diet for 26 weeks. In

p53þ/þ medium-dose mice, relative food consumption was signifi-

cantly higher than in respective controls from weeks 6 to 11; in

p53þ/þ high-dose mice, relative food consumption was significantly

higher than in respective controls in week 3 and weeks 4 to 26 (A). In

p53þ/� medium-dose mice, relative food consumption was higher

than in respective controls in weeks 3, 4, 6, 10, 11, 14 to 17, 19, and

21 to 23. In p53þ/� high-dose mice, relative food consumption was

significantly higher than in respective controls from weeks 1 to 2 (B).

Vol. XX, No. X, 2015 OCHRATOXIN A EFFECTS ON P53þ/� MICE 7

severe. Figure 6 demonstrates a prominent needle-shaped crys-

tal visible in a bronchiole from a control p53þ/� mouse. The

diagnosis of ECP was verified by consultation with the Armed

Forces Institute of Pathology (AFIP 2008), although the

‘‘crystalline’’ component was not considered striking. A fur-

ther observation in the lungs of all mice, regardless of strain

and dose, was that of mild bronchiolar epithelial cell hyper-

plasia, mild smooth muscle hyperplasia of bronchioles and

prominent smooth muscle hyperplasia in the tunica media

of lung arterioles. The degree of smooth muscle hyperplasia

was similar across dose groups and strains. These lesions were

also verified by the AFIP (2008), and are not known to be

associated with ECP.

Immunohistochemical Assessment of Kidneys from OTA-

exposed Mice

Quantitative and semiquantitative immunohistochemical

assessments of kidney sections from p53þ/þ and p53þ/�mice are summarized in Table 5. A dose-dependent increase

in PCNA-positive epithelial cells in the proximal tubules of

p53þ/þ and p53þ/� mice was observed, which was signifi-

cantly greater in both strains exposed to 10-mg OTA/kg diet

compared to the corresponding controls. The number of apop-

totic epithelial cells was also significantly higher in proximal

tubules of p53þ/þ and p53þ/� mice in the 10-mg OTA/kg

dose group. Expression of the kidney injury biomarkers CLU

and OPN in proximal tubules was significantly increased

in p53þ/þ mice in the 10-mg OTA/kg dose group and in

p53þ/� mice in the 2- and 10-mg OTA/kg dose groups

(Table 5; Figure 7). CLU and OPN expression were also higher

with increasing OTA exposure in distal convoluted tubules

from p53þ/þ and p53þ/� mice, with the difference achieving

significance at the highest dose (data not shown).

Comparison of Changes in p53þ/þ and p53þ/� Mice

Differences in the responses of p53þ/� versus p53þ/þmice to OTA were not pronounced. Final BWs were 15% lower

in high-dose p53þ/�mice compared to p53þ/þmice; this dif-

ference was not statistically significant (Table 1). In compari-

son, final BWs were 5%, 0%, and 7% lower in p53þ/� mice

compared to p53þ/þ in the control-, low-, and medium-dose

groups (Table 1). Compared to their respective controls, signif-

icant differences in BW and relative food consumption

occurred more often in p53þ/� than p53þ/þ mice (Figure

2A and B; Figure 3A and B). There were significantly higher

numbers of apoptotic cells in proximal tubules of p53þ/�mice

exposed to 0.5-mg and 10-mg OTA in diet, respectively, com-

pared to p53þ/þ mice exposed to the same levels of OTA in

diet (Table 5). Although CLU and OPN expression were signif-

icantly higher in p53þ/� mice but not p53þ/þ mice exposed

to 2-mg OTA/kg diet, the apparent difference between strains

was not statistically significant. There were no further statisti-

cally significant strain-related differences in responses to OTA

for any other endpoint examined.

DISCUSSION

Hepatic and renal tumors were observed in male B6C3F1

mice exposed chronically to 40-mg OTA/kg diet (Bendele

et al. 1985; Kanisawa and Suzuki 1978). In this study, male

p53þ/þ and p53þ/� mice could not be fed diets containing

15- or 40-mg/kg OTA due to rapid and significant weight loss.

Mice were switched to new diets containing 0.5-, 2-, and 10-mg

OTA/kg diet to replace diets containing 1-, 15-, and 40-mg

OTA/kg diet, respectively. Feed consumption measurements

during the initial period were highly variable in the 15- and

40-mg/kg dose groups, primarily due to the mice removing diet

from feed containers. This activity, presumably motivated by

dissatisfaction with the diet, obscured any measurement of

putative changes in food consumption. However, even with

an assumption that BW changes were due to reduced feed con-

sumption, it could not be established whether feed refusal was

due to unpalatability or to OTA morbidity with concurrent

inappetance.

At the end of the study, a dose-dependent increase in hepa-

tocellular necrosis, but no tumors or preneoplastic lesions were

observed in the livers of p53þ/þ and p53þ/� mice. Likewise,

renal changes were consistent with OTA-induced nephrotoxi-

city, but no tumors were observed in the kidneys of p53þ/þand p53þ/� mice. It is possible that tumors may have devel-

oped if the exposure period had been longer or if it were

possible to use the higher dietary OTA levels that caused

tumors in B6C3F1 mice (Bendele et al. 1985). Validation of the

p53þ/� mouse model for carcinogenesis bioassays initially

indicated that a 26-week exposure period was sufficient for true

positive responses to develop in p53þ/�mice after exposure to

genotoxic compounds known to pose carcinogenic risks for

FIGURE 4.—Pokeweed mitogen (PWM)-stimulated proliferation in

splenocyte cultures from p53þ/þ and p53þ/� mice exposed to ochra-

toxin A in diet for 26 weeks. Proliferation is expressed as 3H-thymidine

incorporation (disintegrations per minute) in PWM-stimulated cul-

tures/3H-thymidine incorporation in unstimulated cultures. *Signifi-

cantly different from corresponding controls (p < .05).

8 BONDY ET AL. TOXICOLOGIC PATHOLOGY

humans (Floyd et al. 2002); however, exposure periods up to 9

months have been used with no increased mortality in control

animals (NTP 2005a, 2005b). In this study, it was assumed that

the maximum tolerable dose had been achieved at 10-mg OTA/

kg diet based on significant weight loss in mice initially

exposed to 15-mg OTA/kg diet. Furthermore, BW and liver

weight reduction in p53þ/� and p53þ/þ mice exposed to

10-mg OTA/kg diet were indicative of morbidity. Final BWs

were 30% and 37% lower, respectively, and relative liver

weights were 27% and 23% lower, respectively, in high-dose

p53þ/þ and p53þ/� mice in this dose group. In comparison,

BWs of B6C3F1 mice exposed to 40 ppm OTA in diet were

20% and 30% lower than those exposed to 0 ppm or 1 ppm

OTA in diet in months 6 to 24 of a 2-year study (Bendele

et al. 1985). This supports the conclusion that the absence of

tumors in p53þ/�mice in this study was not an artifact of dose

selection or exposure period. Since p53þ/� mice are sensitive

to genotoxic carcinogens, the lack of tumors and the overall

similarity in responses to OTA in p53þ/� and p53þ/þ mice

suggest that the MOA for OTA carcinogenesis is primarily

nongenotoxic.

Similar responses to OTA in p53þ/þ and p53þ/� mice

indicate that p53 heterozygosity did not strongly influence

renal toxicity, suggesting either a limited role for p53 and

p53-regulated pathways in response to OTA-induced cellular

toxicity or significant redundancy in cellular protective

mechanisms to compensate for p53 deficiency. The only statis-

tically significant difference between p53þ/� and p53þ/þmice was a higher number of apoptotic cells in kidney OSOM

in p53þ/� versus p53þ/þ mice. The overall difference was

small, but it was consistent with an increase in the incidence

of OTA-induced apoptosis in kidney OSOM and cortex of

p53-deficient gpt d mice compared to their p53-proficient

counterparts exposed to 1- or 5-mg OTA/kg BW by gavage for

4 weeks (Hibi et al. 2013b). Global gene expression analyses of

the kidney outer medulla of gpt d rats exposed to OTA demon-

strated that there were changes in the expression of genes reg-

ulating p53-dependent and p53-independent apoptosis (Hibi

et al. 2013a, 2013b). This study indicates that OTA-induced

apoptosis is likely to be mediated in part by 1 or more p53-

independent pathways. Furthermore, since p53 activates both

pro- and antiapoptotic genes (Roos and Kaina 2013), reduced

expression of p53-dependent antiapoptotic genes associated

with DNA repair may also contribute to the higher incidence

of apoptotic cells in kidneys of p53-deficient mice exposed

to OTA.

TABLE 4.—Summary of severity and incidence of histologic lesions in kidneys from p53þ/þ and p53þ/� mice exposed to dietary OTA for 26

weeks.

Renal Lesion and Location

OTA Added to Diet (mg OTA/kg Diet, Weeks 3–26)

p53þ/þ p53þ/�

0 0.5 2 10 0 0.5 2 10

Apoptosis OSOMa 0.0 + 0.0b 0.1 + 0.3 0.7 + 0.5 2.4 + 0.7d 0.0 + 0.0 0.0 + 0.0 0.3 + 0.7 2.5 + 0.5d

0/10b 1/10 6/9c 9/9c 0/10 0/10 2/10 10/10

MR 0.0 + 0.0 0.0 + 0.0 1.3 + 0.7d 2.7 + 0.5d 0.0 + 0.0 0.0 + 0.0 0.7 + 0.8 2.8 + 0.4d

0/10 0/10 8/9 9/9 0/10 0/10 5/10 10/10

CL 0.0 + 0.0 0.0 + 0.0 0.8 + 0.8 2.0 + 0.0d 0.0 + 0.0 0.0 + 0.0 2.0 + 0.5d 2.3 + 0.5d

0/10 0/10 5/9 9/9 0/10 0/10 10/10 10/10

Karyomegaly OSOM 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 2.4 + 0.9d 0.0 + 0.0 0.0 + 0.0 0.4 + 0.5 2.7 + 0.8d

0/10 0/10 0/9 9/9 0/10 0/10 4/10 10/10

MR 0.0 + 0.0 0.0 + 0.0 1.4 + 0.7 3.4 + 0.5d 0.0 + 0.0 0.0 + 0.0 1.1 + 0.7 3.0 + 0.7d

0/10 0/10 8/9 9/9 0/10 0/10 8/10 10/10

CL 0.0 + 0.0 0.0 + 0.0 1.4 + 0.7d 2.0 + 0.5d 0.0 + 0.0 0.0 + 0.0 1.5 + 1.0d 2.3 + 0.5d

0/10 0/10 8/9 9/9 0/10 0/10 8/10 10/10

Tubular degeneration OSOM 0.0 + 0.0 0.1 + 0.3 0.2 + 0.7 3.0 + 0.0d 0.0 + 0.0 0.1 + 0.3 0.0 + 0.0 3.0 + 0.0d

0/10 1/10 1/9 9/9 0/10 1/10 0/10 10/10

MR 0.0 + 0.0 0.0 + 0.0 1.3 + 1.0 3.0 + 0.0d 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 3.0 + 0.0d

0/10 0/10 6/9 9/9 0/10 0/10 0/10 10/10

CL 0.0 + 0.0 0.1 + 0.3 1.0 + 0.9 3.0 + 0.0d 0.0 + 0.0 0.0 + 0.0 1.4 + 0.7d 2.9 + 0.6d

0/10 1/10 6/9 9/9 0/10 0/10 9/10 10/10

Anisokaryosis OSOM 0.0 + 0.0 0.6 + 0.5 1.0 + 0.5 3.7 + 0.5d 0.0 + 0.0 0.2 + 0.4 1.3 + 0.7d 4.0 + 0.0d

0/10 4/10 8/9 9/9 0/10 2/10 9/10 10/10

MR 0.0 + 0.0 0.1 + 0.3 2.0 + 0.0d 3.8 + 0.4d 0.0 + 0.0 0.2 + 0.4 2.0 + 0.8d 3.7 + 0.5d

0/10 1/10 9/9 9/9 0/10 1/10 9/10 10/10

CL 0.0 + 0.0 0.2 + 0.4 1.3 + 1.0 2.1 + 0.9d 0.0 + 0.0 0.2 + 0.4 2.1 + 0.9d 3.0 + 0.5d

0/10 2/10 6/9 8/9 0/10 2/10 9/10 10/10

aOSOM ¼ outer stripe of outer medulla; MR ¼ medullary rays; CL ¼ cortical labyrinth. bSeverity data are expressed as mean + SD for lesion severity scores from 0 to 5, where

increasing scores are indicative of increasing lesion severity. Incidence data are expressed as # affected animals/total number animals per group. cHydronephrosis was observed in one

kidney in the medium-dose group and no further morphological changes were assessed; one kidney in the high-dose group was compromised at collection and could not be assessed.dLesion severity was significantly different from corresponding controls using analysis of variance (ANOVA) on ranks followed by post hoc pairwise comparisons using the Dunn’s or

Tukey test (p � .05).

Vol. XX, No. X, 2015 OCHRATOXIN A EFFECTS ON P53þ/� MICE 9

OTA stimulates and disrupts cell division in the kidney. A

dose-dependent increase in PCNA-positive cells, indicative

of proliferation, was observed in proximal tubules of p53þ/þand p53þ/� mice and was consistent with similar increases

in PCNA-positive proximal tubule cells in rats exposed to OTA

by gavage (Mally et al. 2005; Taniai et al. 2014). The presence

of cells with an abnormally enlarged nucleus, or karyomegaly,

in the renal proximal tubule is a distinguishing lesion associ-

ated with OTA exposure (Boorman et al. 1992). Although kar-

yomegaly can be induced by renal carcinogens, it is not

considered a preneoplastic lesion because there is no evidence

that proliferative foci stem directly from these cells (Hard et al.

1999). OTA and other karyomegaly-inducing carcinogens,

which also stimulated cellular proliferation, increased the

expression of gH2AX, a phosphorylated variant of the histone

H2AX involved in DNA double strand break (DSB) repair, in

the kidney OSOM of rats exposed to 210-mg OTA/kg bw/d

by gavage for 28 days (Taniai et al. 2012a). Phosphorylated

Chk2 and nuclear cdc2, both part of the G2/M DNA damage

checkpoint pathway, were also elevated suggesting that DNA

damage is associated with exposure to karyomegaly-inducing

carcinogens, triggering homeostatic repair responses that pre-

vent cells with DNA damage from entering mitosis. Multiple

epigenetic effects resulting in cell cycle perturbation have been

linked experimentally to OTA toxicity and carcinogenicity,

including aberrant ubiquitin D activation leading to disrupted

cell cycle regulation, inhibition of histone aceytltransferases

leading to mitotic disruption, and cytoskeletal perturbations

leading to aberrant cell division (Taniai et al. 2012b; Czakai

et al. 2011; Rached et al. 2006). One or more of these events

leading to mitotic disruption in renal proximal tubule cells

could manifest as OTA-induced karyomegaly. The hypothesis

that OTA-induced aberrant mitosis is a trigger for proliferative

changes in renal proximal tubules is plausible (Rached et al.

2007; Mally 2012), but the key event has yet to be conclusively

confirmed and the available evidence does not rule out multiple

cellular targets for OTA. Marin-Kuan et al. (2008) proposed

that OTA carcinogenicity stems from multiple epigenetic

mechanisms, including cell- and tissue-level responses to

OTA-meditated apoptosis, and tissue regeneration and prolif-

eration resulting from cytotoxicity due to oxidative stress and

protein synthesis inhibition. This hypothesis accommodates the

spectrum of cellular changes observed in kidneys from OTA-

exposed p53þ/þ and p53þ/� mice that ranged from necrosis

FIGURE 5.—Photomicrographs of kidney tissues from control mice and

mice exposed to ochratoxin A (OTA) in diet: (A) kidney cortex from a

p53þ/� control mouse; (B) kidney cortex from a p53þ/� mouse

exposed to 10-mg OTA/kg diet. In (B), the large black arrowheads

indicate proximal convoluted tubular epithelial cell with karyome-

galy, the white arrowheads indicate apoptotic bodies, and the arrows

indicate damaged tubules containing vacuolated epithelial cells.

Hematoxylin and eosin stain; scale bars 50 mm.

FIGURE 6.—Photomicrograph of a lung section from a p53þ/� control

mouse; hematoxylin and eosin stain. A large needle-shaped crystal is

evident in the bronchiole (black arrowhead); in the adjacent alveoli, a

light mixed leukocytic infiltration composed of macrophages with

acidophilic cytoplasm and a few neutrophils is evident. Hematoxylin

and eosin stain; scale bar 100 mm.

10 BONDY ET AL. TOXICOLOGIC PATHOLOGY

TABLE 5.—Immunohistochemical assessment of kidneys from p53þ/þ and p53þ/� mice exposed to dietary OTA for 26 weeks.

Endpointa

OTA Added to Diet (mg OTA/kg diet, Weeks 3–26)

p53þ/þ p53þ/�

0 0.5 2 10 0 0.5 2 10

# PCNA positive cells 385.6 + 55.3 441.5 + 122.7 519.0 + 31.3 738.8 + 93.4b 389.9 + 41.7 478.1 + 48.6 455.0 + 105.6 799.2 + 83.7b

# Apoptotic cells 453.3 + 40.7 453.3 + 75.8 551.0 + 27.4 729.6 + 55.6b 471.1 + 47.3 499.1 + 22.7c 550.9 + 24.7 817.2 + 83.4b,c

Clusterin expression 18.5 + 6.7 29.0 + 16.1 33.3 + 15.4 47.5 + 12.7b 21.6 + 8.1 34.5 + 13.8 40.5 + 16.9b 50.0 + 7.5b

Osteopontin expression 27.0 + 9.5 42.5 + 17.5 53.9 + 12.4 75.5 + 6.0b 30.5 + 10.9 51.0 + 14.9 59.0 + 13.7b 81.0 + 8.4b

aData are expressed as meanþ SD of # proliferating cell nuclear antigen (PCNA) positive/mm2 cells or # apoptotic cells/mm2 in proximal tubules of 1 kidney section/mouse. Clusterin

and osteopontin expression data are expressed as mean þ SD of semiquantitative percentage positivity scores for proximal tubule staining on 1 kidney section/mouse as described in the

Methods and Materials. For each endpoint, 1 kidney section was assessed for a total n ¼ 9 or 10 mice/dose/strain (as in Table 4). bData are significantly different from corresponding

controls using analysis of variance (ANOVA) on Ranks followed by post hoc pairwise comparisons using the Dunn’s test (p � .05). cData are significantly different from corresponding

p53þ/þ mice using 2-way ANOVA on Ranks followed by post hoc pairwise comparisons using the Dunn’s test (p � .05).

FIGURE 7.—Clusterin (CLU) and osteopontin (OPN) expression in the outer stripe of the outer medulla of kidneys from p53þ/þ mice: (A)

CLU, control; (B) CLU, 10-mg ochratoxin A (OTA)/kg diet; (C) OPN, control; (D) OPN, 10-mg OTA/kg diet. CLU and OPN positivity are

visible as dark reddish-brown staining, more prominent in kidney sections from the 10-mg OTA/kg dose group. Scale bars 100 mm.

Vol. XX, No. X, 2015 OCHRATOXIN A EFFECTS ON P53þ/� MICE 11

and apoptosis to proliferation and changes such as karyome-

galy, anisokaryosis, and hyperchromatic nuclei in renal epithe-

lial cells.

Based on experimental evidence of OTA carcinogenicity in

rats, mice, and poultry (Bendele et al. 1985; NTP 1989; Stoev

2010), DNA damage is a likely outcome of OTA exposure. In

confirmation, in vivo mutagenesis assays using gpt d rats

detected large deletion mutations in the outer medulla of the

kidney after 4 weeks of exposure to 5-mg/kg OTA by gavage

(Hibi et al. 2011). Positive comet assays and increased phos-

phorylation of histone variant H2AX were indicative of DNA

DSBs at the target site and increased expression of genes linked

to DNA repair by homologous recombination (HR) suggested

that large deletion mutations may arise during repair by HR

of OTA-induced DSBs (Kuroda et al. 2014). However, the

underlying mechanism of OTA-induced DSBs has yet to be

determined. Nuclear 8-hydroxy-20-deoxyguanosine (8-OHdG)

levels were not altered in the cortex or outer medulla of gpt drats exposed to OTA, suggesting that oxidative DNA damage

was not the primary cause of deletion mutations at the target

site in this model (Hibi et al. 2011), but other epigenetic path-

ways leading to OTA-induced genotoxicity cannot be ruled out

(Mally and Dekant 2009; Trosko and Ruch 1998).

Changes in CLU and OPN expression in kidneys from

p53þ/þ and p53þ/� mice were similar to those seen in kid-

neys from OTA-exposed rats (Rached et al. 2008). Dose-

dependent increases in CLU and OPN positivity in proximal

and distal tubules due to OTA exposure in mice were consistent

with their identification as presumptive biomarkers of both

proximal and distal tubule injury (Hoffmann et al., 2010).

Although CLU and OPN have been used as renal injury mar-

kers, both are widely distributed in tissues. The secretory het-

erodimeric glycoprotein CLU has been reported to be

involved in regulating multiple cellular responses that may

occur in a mutually exclusive and opposing manner, for exam-

ple, apoptotic cell death, cell cycle regulation, DNA repair, cell

adhesion, tissue remodeling, lipid transportation, membrane

recycling, and immune system regulation (Jones and Jomary

2002; Shannan et al. 2006). OPN, a secretory glycosylated

phosphoprotein, is an antiapoptotic factor associated with reg-

ulation of bone remodeling, chemotaxis, and immune

responses, as well as cellular proliferation and regeneration

after renal injury (Xie et al. 2001). Consequently, increased

renal CLU and OPN expression in OTA-exposed mice are con-

sistent with an increased incidence of proliferating cells, apop-

totic cells, necrosis, and other lesions indicative of cell cycle

disruption.

There were no significant changes in peripheral and splenic

WBC numbers, including granulocyte differentials and lym-

phocyte subsets, in p53þ/þ and p53þ/� mice chronically

exposed to OTA. Similarly, there were no significant changes

in peripheral WBC numbers in rats chronically exposed to

OTA in diet (NTP 1989). Mild splenomegaly in all high-dose

p53þ/þ and p53þ/� mice was most likely a nonspecific

response to tissue damage in target organs such as the kidney.

Cellular proliferation was significantly depressed in ex vivo

splenocyte cultures indicating functional compromise in lym-

phocytes from OTA-exposed mice, which was generally in

accordance with the well-established immunosuppressive

effects of OTA (Bondy and Pestka 2000).

The lung lesions observed in all p53þ/� and p53þ/þ mice

were unexpected but not considered to be treatment related.

ECP is a sporadic, idiopathic disease that occurs in certain

strains of laboratory mice, including the C57BL/6 and 129Sv

strains used to generate this p53þ/� knockout and the reconsti-

tuted p53þ/þ mouse (French, Storer, and Donehower 2001b;

Hoenerhoff, Starost, and Ward 2006). To our knowledge, this

is the first report of ECP in this mouse model. The disease was

considered mild, but there was apparent exacerbation with

OTA treatment resulting in a slight increase in the severity of

ECP in high-dose mice. Since ECP has been associated with

mutations that result in immunodeficiency or that target the

immune system (Guo, Johnson, and Schuh 2000), it is possible

that OTA-induced immunosuppression contributed to

increased ECP severity. The pulmonary smooth muscle hyper-

plasia that was also observed in all mice is unexplained at this

time but was not considered to be treatment related and was not

increased in severity with OTA exposure.

OTA is nephrotoxic to laboratory and domestic animals and,

irrespective of its MOA, is considered a potential human health

hazard. The lowest observed effect level (LOEL) for renal toxi-

city in the pig, which is highly sensitive to OTA-induced

nephrotoxicity, was 8-mg/kg BW/day for minimal renal toxicity

(JECFA 2008; Krogh et al. 1974). The LOEL for renal proxi-

mal tubule karyomegaly in male rats exposed to OTA by

gavage for 5 d/wk for 90 d was 62.5 mg/kg BW/d (NTP

1989). In 2-year carcinogenesis studies the LOEL for kidney

tumors was 4,400 mg/kg BW/d in male B6C3F1 mice exposed

to OTA in diet, and 70 mg/kg BW/d in male rats exposed to

OTA by gavage for 5 d/wk (Bendele et al. 1985; JECFA

2008; NTP 1989). In comparison, the lowest dose group in

which renal changes were observed in p53þ/� and p53þ/þmice was 2-mg OTA/kg diet, corresponding to a LOEL of

200-mg OTA/kg BW/day based on feed consumption. This is

lower than the LOEL for kidney tumors in B6C3F1 mice,

although the wide dose separation between the 1 ppm and 40

ppm dietary OTA levels in the 2-year mouse study complicates

direct comparison with the present study. Taken together, these

comparisons support the general conclusion that mice are less

sensitive than rats and pigs to OTA-induced nephrotoxicity and

less sensitive than rats to OTA-induced renal carcinogenesis.

Species sensitivity was arguably a more critical influence on

responses to OTA than p53 heterozygosity.

In conclusion, renal changes due to ochratoxicosis in

p53þ/þ and p53þ/�mice were consistent with those observed

in B6C3F1 mice and in other species. Heterozygosity for the

p53 gene had a limited influence on murine responses to OTA.

The results of this study were interpreted to be suggestive of

primarily epigenetic MOAs for OTA carcinogenesis, based

on validation of the p53þ/� N5 mouse for use in short-term

carcinogenesis bioassays which has shown that this model is

susceptible to tumor induction by genotoxic and not

12 BONDY ET AL. TOXICOLOGIC PATHOLOGY

nongenotoxic carcinogens (Pritchard et al. 2003; Tennant,

Spalding, and French 1996).

ACKNOWLEDGMENTS

The authors would like to thank Jennifer Eastwood, Mark

Feeley, Gary Lombaert, and Tim Schrader for valuable discus-

sions and article review, and the staff of the Health Canada

animal care facility for their assistance throughout the study.

AUTHOR CONTRIBUTION

Authors contributed to conception or design (GB, LC, IC,

DL, and RM); data acquisition, analysis, or interpretation

(GB, DC, SA, LC, CA, IC, RK, KK, DL, and RM); drafting the

manuscript (GB); critically revising the manuscript (DC, SA,

LC, CA, DL, and RM); and gave final approval (RM). All

authors gave final approval, all authors agreed to be accounta-

ble for all aspects of work in ensuring that questions relating to

the accuracy or integrity of any part of the work are appropri-

ately investigated and resolved.

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