Mechanistic studies on genotoxicity and carcinogenicity of salicylazosulfapyridine an anti-inflammatory medicine

Exp Toxic Patho11997 ; 49: 15-28 Gustav Fischer Verlag

lAmerican Health Foundation, New York 2National Institute of Environmental Health Sciences, North Carolina 3National Center for Toxicological Research, Arkansas, USA

Mechanistic studies on genotoxicity and carcinogenicity of salicylazosulfapyridine an anti-inflammatory medicine MICHAEL J. IATROPOULOS 1, GARY M. WILLIAMS i , KAMAL M. ABDO\ FRANK W. KARI 2 , RONALD W. HART 3 With 1 figure and 7 tables Received: July 2, 1996; Accepted: August 4, 1996 Address for correspondence: Dr. M. J. Iatropoulos, American Health Foundation, One Dana Road, Valhalla, NY 10595, USA. Key words: Genotoxicity; Carcinogenicity; Salicylazosulfapyridine (SASP); Toxicity testing methodology; Risk assessment; Rodent human extrapolation.

Summary Salicylazosulfapyridine (SASP), which has been in clinical use for over 50 years, was reported by the National Toxicology Program to increase rat (F344 strain) urinary bladder and mouse (B6C3F 1 hybrid) liver tumors under ad libitum (AL) feeding conditions, while under a feed restriction (FR) regimen, these tumors were not increased. The present investigations were undertaken to assess the implications of these results for the safety of SASP in humans. SASP and its 2 major metabolites, 5-aminosalicylic acid (ASA) and sulfapyridine (SP) were tested for in vivo induction of micronuclei in mouse bone marrow cells with or without prefolic treatment and for in vivo formation of DNA adducts in rat and mouse liver and urinary bladder. None exhibited mutagenicity or DNA reactivity. SASP and SP have induced sister chromatid exchanges and micronuclei (MN) in cultured human lymphocytes in the absence of liver activation enzymes and in B6C3F 1 mice (but not in rats) MN in bone marrow and peripheral RBC. Treatment with folate reduces the frequency of MN. Perhaps the short (28 days) RBC lifespan in mouse underlies the sensitivity of this species. Thus, SASP without folate supplementation is an aneuploidogen. In a 2-year study in AL fed SASP-treated (high dose 337.5 mg/kg) rats, urinary pH was increased and urinary specific gravity was reduced at 60 weeks. At the end, this SASP group showed urothelial hyperplasia and papillomas in the urinary bladders of male rats primarily. In the FR high dose SASP group, the hyperplasia was reduced from 82 % to 14 %. At the end of 2 years, the incidence of multiorgan leukemia was reduced in both AL and FR high dose

SASP groups. Thus, SASP caused intraluminal bladder changes in the rat (especially males) consisting of chronic urothelial stimulation, concretions, hyperplasia which resulted in neoplasia. In the mouse, because of species differences in liver ratios (mouse> rat) and, increasing (3 times higher) liver perfusion in the mouse, compared to the rat, there was hepatocellular toxicity and resulting preneoplasia and neoplasia within 2 years. These findings occurred in all AL SASP groups (flat curve without dose response); but were reduced under FR conditions. In this species, the multiorgan lymphoma incidence was reduced in both AL and FR high dose SASP groups. Thus, SASP and its major metabolites are not genotoxic. Folate deficiency associated with SASP administration is probably responsible for aneuploidy in lymphocytes and erythrocytes. SASP does not induce neoplasia directly in either livers, urinary bladders or other organs. Accordingly, SASP is judged to pose no carcinogenic risk to humans.

Introduction Salicylazosulfapyridine (SASP) has been in clinical use for more than 50 years and has been extensively used for the treatment of ulcerative colitis, Crohn's disease (BADLEY 1975; CAPRILLI et al. 1975; GARDNER 1976), rheumatoid arthritis (MCCONKEY et al. 1980; BIRD et al. 1982), and psoriatic arthritis (FARR et al. 1990). To date, more than lO million patient-years have been accrued in patients benefitting from both short and long-term Exp Toxic Pathol49 (1997) 1-2

15

therapy for inflammatory bowel diseases (PINCZOWSKI et al. 1994). The average usual maintenance dose is 2 grams per day and the average treatment length is at least 3 months (DAVIES and RHODES 1978; Rns et al. 1979). Patients with ulcerative colitis generally receive SASP treatment for the longest times. This disease results in an increased risk for colorectal cancer (PRIOR et al. 1982; GYDE et al. 1988; EKBOM et al. 1990), possibly because of increased levels of cell proliferation. Folate deficiency has been implicated as a risk factor in colorectal neoplasia of ulcerative colitis patients (LASHNER et al. 1989). SASP was found to cause impaired folate absorption by inhibiting its transport across the intestine and by inhibiting folate metabolizing enzymes (FRANKLIN and RoSENBERG 1973; SELHUB et al. 1978). Consequently, it was suspected that SASP could increase the risk of colorectal neoplasia in patients with ulcerative colitis. In a well controlled study of ulcerative colitis patients given SASP with or without folate supplementation, the risk of colorectal neoplasia was decreased rather than increased (LASHNER et al. 1989). PINCZOWSKI et al. (1994) also reported a significant reduction in the incidence of colorectal cancer associated with SASP therapy in a cohort of 3112 ulcerative colitis patients. ALSO, ODES and FRASER (1989) have shown that one possible explanation for the low incidence of colorectal cancer among ulcerative colitis patients was the sustained maintenance therapy with SASP. Clearly, the presence of untreated chronic colorectal inflammation is probably implicated in the increased risk of colorectal neoplasia. SASP has been shown to inhibit several inflammatory cell functions, including superoxide production (ALI et al. 1982; GIBSON and JEWELL 1985; BETTS et al. 1985; COMER and JASIN 1988; CARLIN et al. 1989). Furthermore, SASP and its main metabolites inhibit bacterial growth in the colon (SANDBERG-GERTZEN et al. 1985). In experimental studies, SASP reduced the extent and severity of dimethylhydrazine (DMH) induced colorectal neoplasia in rats (ADRIANOPOULOS et al. 1989). On the other hand, because of its effect on folate, SASP was implicated as a co-carcinogen in the same rat DMH model (DAVIS et al. 1992). After a debate (MEEHAN 1993), these findings were reinterpreted by the original authors to indicate that SASP was not a cocarcinogen. In all species, including man, orally administered SASP undergoes azoreductive cleavage by the colonic bacterial flora and is bioconverted to 5-aminosalicylic acid (ASA) and sulfapyridine (SP). ASA is the therapeutically active metabolite. SASP shows inhibitory activity on the production of cytokines such as IL-2 (FUJIWARA et al. 1990a, 1990b) and IL-l (REMVIG AND ANDERSEN 1990) and, in general, inhibits superoxide production by inflammatory cells (CARLIN et al. 1989). SASP is a powerful scavenger of oxygen radicals (BETTS et al. 1985; CARLIN et al. 1989). Also, SASP is an inhibitor of prostaglandin 15-hydroxydehydrogenase as well as glutathione transferase (BACH et al. 1985) and xanthine oxidase (CARLIN et al. 1985). 16

Exp Toxic Pathol49 (1997) 1-2

SASP is poorly absorbed from the intestines of mice, rats and man. Both rats and mice clear the parent compound to a high extent in the bile (unlike man), and thus achieve comparatively low plasma concentrations. In plasma, SASP is highly bound (99 %) to serum albumin in all species. Likewise, the metabolism is similar in all species, although rodents display more metabolites than parent compound. Rats eliminate SASP more slowly than mice (PEPPERCORN and GOLDMAN 1972; CHUNGI et al. 1989; ZHENG et al. 1993; TETT 1993, NTP 1994). SASP and its two main metabolites SP and ASA were reported negative in Salmonella typhimurium gene (point) mutation assays, either with or without induced mouse or rat liver S9 enzymes (VOOGD et al. 1980; ZEIGER et al. 1988). Neither SASP (BIOSHOP et al. 1990) nor ASA (WITT et al. 1992a) induced increases in sister chromatid exchanges (SCE) or chromosomal aberrations (CA) in cultured Chinese hamster ovary cells (CHO), either with or without rat liver S9. In CHO cells, SP exposure resulted in a small but significant increase in SCE in the absence of S9, but did not induce CA, either with or without rat liver S9 (WITT et al. 1992a). SASP induced SCE and micronuclei (MN) in cultured human lymphocytes at the same concentrations that yielded negative results with CHO cells in the absence of S9 (MACKAyet al. 1989). In the same assay, SP induced only SCE, and ASA induced neither SCE nor MN. SASP has been reported to induce increases in the frequency of SCE and MN in lymphocytes obtained from patients receiving chronic SASP therapy, although considerable interindividual variability in MN, as well as exposure to concomitant medications were complicating factors (Fox et al. 1987). SASP and SP induced increased frequency of MN of peripheral RBCs in mice dosed by gavage for 90 days (BIOSHOP et al. 1990), and increased MN of bone marrow cells in mice dosed by gavage for 2-3 days «BIOSHOP et al. 1990; WITT et al. 1992b). SASP was positive in a micronucleus assay conducted with peripheral blood from mice on a 13-week gavage study at 675, 1350 or 2700 mg/kg/day (MACGREGOR et al. 1990). The majority of micronucleated RBCs were shown to contain kinetochores, which indicates that in the MN there was a failure of mitotic chromosomal segregation (WITT et al. 1992b). SASP was assessed for CA in male and female mouse bone marrow (femur) in vivo at 1000, 2000 or 4000 mg/kg administered once daily for up to 3 days. Each dose was separated by a 24-hour interval. Significant increases in micronucleated polychromatophilic erythrocytes (PCEs) were observed in both genders (NTP 1995a). SASP was also assessed for CA in male rat bone marrow from 337.5 to 3000 mg/kg in 2 separate trials. SASP induced a non dose-related but significant increase in the frequency of PCEs in the high dose (2700 mg/kg) of trial 1, but negative results in trial 2 at a higher dose (3000 mg/kg) (NTP 1995a). Finally SASP from 1000 to 4000 mg/kg was also given daily for 3 days to male mice, without inducing CA in bone marrow cells (NTP 1995a).

Table 1. Experimental design of multiple dose studies with SASP in rats and mice. Species and Gender

Animals per group and gender

Dose Levels (mg/kg/day)

Dose range finding studies Rats M + F 0-84-168.8-337.5 10 MiceM +F 10 0-675-1350-2700 Carcinogenicity studies Rats M + F 0-84-168-337.5 60 (l0)" Rats M 70 337.5 (lot o (weight matched)d-337.5 Rats M 50 o (feed restricted)e -337.5 Rats M 50 Mice M + F 60 0-675-1350-2700 (l0)" o (weight matched)d -2700 MiceM 50 MiceM 50 o (feed restricted)" -2700 DNA adduct study 41 Rats M 0-300-1000 MiceM 41 0-1000-3000 Micronucleus study (with prefolic) 109 Mice M 0-1250-2500-5000Prefolic 50-1250 + 502500 + 50-5000 + 50

Duration (weeks)

Interim Sacrifices (weeks)

Recovery (weeks)

none none

none none

5

103M 104F 103 b

60 a 60a 26 c.60c

none none 77 b

5

103 103 103M 104F 103 t03

none none 60a 60 a none none

none none none none none none

5 5

none none

none none

2 2

none

none

10

13 13

0.5/l .5g

Housing (no/cage)

5

10 Male (M) and 10 female rats and mice from each group were evaluated at 60 weeks (15 months). Exposed only for 26 weeks and were subsequently on recovery for 77 weeks. c 10 vehicle control and to high dose (337.5) rats were evaluated at 26 and 60 weeks. d The daily feed allotment for this control group was restricted so that the mean body weight matched the mean body weight of the high dose group. e The daily feed offered to control and high dose groups was limited to approximately 85 % of the ad libitum fed control group. f Part of a 32P-postlabeling study capable of detecting adduct formation in rat and mouse liver and urinary bladder DNA; 2-acetylaminofluorene (for liver) and [3-2-naphthylamine (urinary bladder) were used as positive controls; both were administered by gavage. g Part of a mouse bone marrow micronucleus study; prefolic was given i.v. 7 days before and 3 days concomitantly with SASP; cyclophosphamide (i.p.) was used as a positive control. a

b

In summary, SASP was found to be clastogenic only in cultured human lymphocytes. SASP and SP induced aneuploidy in mouse bone marrow RBCs in vivo, with no evidence of mutagenic potential. The U.S. National Toxicology Program conducted two 13-week and two 2-year studies with SASP in F344JN rats and B6C3F 1 mice (NTP 1995a). An outline of these studies is given in table I (NTP 1995a; 1995b; ABDO and KARl 1996). In order to study the effects of dietary restriction, on the incidence of tumors (Ross and BRAS 1973; POLLARD et al. 1984), different feeding groups were added to the two ad libitum fed 2-year studies (males only). These additional groups consisted of weight-matched (WM) groups, in which the non-treated animals were fed in such a way that their mean body weight matched that of the high-dose SASP ad libitum CAL) group. In the diet restricted (DR) group, animals

were given diets limited such that the control group attained body weights of approximately 80 % that of the AL fed controls (ABDO and KARl 1996). The nonneoplastic and neoplastic data from the 2-year rat study are given in tables 2 and 3, and from the 2-year mouse study in tables 4 and 5. In AL-fed rats, urinary bladder papillomas were increased from 0 to 12 % in the high dose (337.5 mg/kg) SASP groups with concomitant reduction (from 26 % to 6 %) of mononuclear cell leukemia (MCL). In the WM (high dose SASP) groups, the neoplastic changes were the same, i.e. the incidence increase (in papillomas) was 12 % and the incidence in MCL was 6 %. In the DR (high dose SASP) groups, no papillomas were present, and the incidence of MCL was 4 % compared to 22 % in the controls. In mice, there was an increase in hepatocellular neoplasia in the AL high dose SASP group (48 % control vs 88 % high dose), and Exp Toxic Pathol 49 (1997) 1-2

17

I' tv

:::3

= = = =

Hyperplasia Dilation F.C. hypo *

VB

= =

Hemosiderin HCP MO

=

0 0 0

0 0 0 0 0 0 0 0 0 30 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 20 0 0 10 0 40 20 0

168,

0 0 26

0 0 0 0 0 0 20 6 28 28 2 0 0 36

2 0 0 2 2 2 20 20 30 30 0 04* 0 24

4 2 28 2 26 2 40 22 30 34 0

SASP-exposed groups similar to control

103 Weeks Male Rats 84, 0,

12* 0 6**

14 20 82 22 66 56 86 26 60 46 2

337.5 168,

0 0 10

0 0 0 0 0 0 0 90 80 40 0 0 0 0

0 0 0 0 0 0 0 100 90 60 0 0 0 10

0 0 0 0 10 0 20 80 80 20 0

High dose group 11 % lower than control 4.5 4.1 4.8 1.029 1.035 1.028

60 Weeks Female Rats 0, 84,

All data were obtained from National Toxicology Program (NTP) Technical Report 457, 1995a. urinalysis includes volume (m1116 hr) and specific gravity (SG). hemosiderin pigmentation present. hematopoietic cell proliferation. mononuclear cell leukemia in multiple organs. urinary bladder. transitional (urothelial) cell hyperplasia in both VB and kidney. in the kidney it refers to renal tubule dilation; in the VB it refers to VB dilation. follicular cell hyperplasia. significantly different at p < 0.05; ** at p < 0.01, from the control group.

0 0 0

Neoplastic effects VB: Papilloma Kidney: Papilloma MO: MC leukemia

b

0 0 0 10 0 0 0 0 20 10 0

Vrinalysis b

"""

.....

\C

'D 'D

~

168, 337.5,

High dose group 7 % lower than control 2.9 2.7 4.3 3.7 1.043 1.045 1.040 1.041

vol SG Nonneoplastic effects VB: Dilation Concretion Hyperplasia Kidney: Dilation Concretion Hydronephrosis Hyperplasia Mineralization Spleen: Hemosiderin HCP Thyroid: F.e. hypo

Body weight changes

2-

60 Weeks Male Rats 0, 84,

Summary of incidence (%) of all pertinent effects in rats in the 2-year gavage study with SAspa.

Duration Gender Dose Groups (mg/kg) Pertinent Effects

Table 2.

S-

'1:1

C=;"

-l 0 ><

"0

><

tTl

00

0 0 0

0 0 20 0 60 10 80 90 100 0 0

7.8 1.026

337.5

0 0 28

0 0 4 4 0 0 6 88 0 0 0

0 0 18

0 0 0 4 18 0 14 94 0 0 0

4* 0 16

0 2 8 2 68 6 46 86 0 0 0

0 4* 6**

0 0 24 12 74 20 86 82 0 0 2

High dose group 2 % lower than control

104 Weeks Female Rats 0, 84, 168, 337.5

Table 3: Summary of incidence (%) of all pertinent effects in rats in the 2-year gavage study with high dose SASpa on three different modes of feed allotment.

Dose (mg/kg)

0

Body Weight changes Nonneoplastic effects UB: Dilation Calculus Concretion Hyperplasia

Restricted-Feed Control d

Weight-Matched Control c

Ad libitum Control b

Mode of Feeding

337.5

0

SASP-exposed groups similar to control

337.5

0

337.5

337.5 group 337.5 group 6 % lower than control 10 % lower than control

0 0 0 0

14 54 20 82

2 0 0 0

14 54 20 82

0 0 0 0

0 0 0 14

Kidney:

Dilation Concretion Hydronephrosis Hyperplasia Mineralizati on

0 0 0 20 6

22 66 56 86 26

2 0 0 10 12

22 66 56 86 26

0 0 0 6 4

0 44 0 36 22

Spleen:

Hemosiderin HCP

28 28

60 46

40 18

60 46

24 0

70 0

0 4 26

12* 0 6**

0 2 20

12* 0 6**

0 4 22

Neoplastic effects UB: Papilloma Alv. Br. ad. Lung: MC leukemia MO:

= = = d

=

Hemosiderin HCP MO UB Hyperplasia Dilation Alv. br. ad. *

= = = = = = = =

0 0 4**

All data were obtained from NTP Technical Reports 457, 1995a and 460, 1995b. Ad libitum control and high (337.5 mg/kg/day) dose groups. The daily feed allotment of the control group was restricted so that its mean body weight matched the one of the high dose group. The daily feed offered to both control and high dose groups was approximately 85 % of the ad lib fed control group. hemosiderin pigmentation present. hematopoietic cell proliferation. mononuclear cell leukemia in multiple organs. urinary bladder. transitional cell (urothelial) hyperplasia, in both UB and kidney. in the kidney in refers to renal tubule dilation; in the UB it refers to UB dilation. alveolar bronchiolar adenoma. significantly different at p < 0.05; ** at p < 0.01, from the control group.

a decrease in malignant lymphomas from 10 % to 2 %. In both WM and DR high dose SASP group, liver tumors were decreased from 28 % to 7 % in WM and from 35 % to 18 % in DR. The decrease in malignant lymphomas was from 9 % to 0 in WM and from 8 % to 0 in DR. The purpose of the present paper is to report recent nonclinical genotoxicity, mechanistic safety assessment and pharmacokinetic studies. Some have been published in part (PuLci et al. 1995; ABDO and KARl 1996; IATROPOULOS et al. 1996). This paper will compare and correlate these studies to other published data, and assess the relevant human genotoxic and carcinogenic risks resulting from chronic therapeutic exposure to SASP.

Materials and methods Test Materials: Sulfasalazine (SASP) or 2-hydroxy-5[[4-[2-(pyridinylamino) su1fonyl]phenyl]benzoic acid in all published and unpublished studies reviewed in this paper was provided by Pharmacia. The vehicle for all the recent studies reviewed in this paper was com oil. The mode of administration was daily gavage. Whenever possible the vehicle for the positive control substances was com oil. Pharmacokinetics Studies: The analytical methods used for both parent compound and metabolites were based on high pressure liquid chromatography with UV detection for SASP and sulfapyridine (SP) metabolites and fluorescence detection for 5-amino-salicylic acid (ASA). In most Exp Toxic Pathol 49 (1997) 1-2

19

Table 4. Summary of incidence (%) of all pertinent effects in mice in the 2-year gavage study with SASpa. Duration Gender Dose Groups (mg/kg) Pertinent Effects

103 Weeks Male Mice 675

Body weight changes

High dose group 12 % lower than control

Nonneoplastic effects Liver: Cytol. alter. C.c. focus E. focus Mixed focus Hemosiderin Spleen: HCP F.C. hypo Thyroid:

o

1350

2700

104 Weeks Female Mice 675

o

1350

2700

High dose group 10 % lower than control

4 12 0 4 22 18

28 38 0 50 32 8

10 40 0 65 41 6

22 44 0 94 26 4

0 10 4 28 14 40

2 34 0 74 42 10

2 30 2 78 38 4

2 39 0 94 47 2

26 26 4 10

64 30 18 6

56 46 26 2**

84** 16 12 2**

24 4 0 26

56* 20 12 12*

50* 20 14 14*

58* 18 16 12*

Neoplastic effects Liver:

MO: Hemosiderin HCP MO Cytol. alter C.c. focus H. ad. H. carc. F.e. hypo *

H. ad. H. carc. H. ad. & carc. Lymphoma = = = = = = = = = =

All data were obtained from National Toxicology Program (NTP) Technical Report 457, 1995a. hemosiderin pigmentation present. hematopoietic cell proliferation. malignant lymphoma in multiple organs. hepatocellular cytologic alterations. clear cell focus ; E. focus = eosinophilic focus. hepatocellular adenoma. hepatocellular carcinoma. follicular cell hyperplasia. significantly different at p < 0.05; ** at p < 0.01, from the control group.

of the studies, radiolabeled SASP was used with the 14C_ label in the ASA part of the molecule and 3H in the SP moiety. Absorption, distribution, metabolism and excretion have been studied in the rat and mouse and reported in internal unpublished company reports (Pharmacia, Sjoqvist, 1989; Pharmacia, Anderson, 1990; Pharmacia d'Argy 1990), as well as published studies (ZHENG et al. 1993).

Genotoxicity Studies: Pharmacia has also conducted four genetic toxicity studies. In the bacterial point mutation assay, Salmonella tester strains T A98 and T A 100 and E. coli WP2p and WP2 uvrA-p were exposed to SASP concentrations ranging from 0.4 to 6250 Ilg/plate, with or without S9. In a mouse lymphoma gene mutation study, SASP at 0.22 to 700 Ilg/ml or 50 to 500 Ilg/ml was tested to determine whether it can induce mutations to 6-thioguanine resistance in mouse lymphoma L51784 cells using a fluctuation assay with or without S9. In an in vivo chromosome aberration study in rat bone marrow, male and female SpragueDawley rats were exposed orally to 500 mg/kg of SASP given once and killed 6,24 and 48 hours later. Subsequently, the chromosome damaging potential of SASP on bone marrow cells was evaluated. Lastly, human lymphocyte cultures were exposed to concentrations of SASP ranging from 20

Exp Toxic Pathol 49 (1997) 1-2

2.5 to 100 Ilg/ml. All these studies were conducted at ToxicollMicrotest Labs, UK.

Folate Supplementation Study: Two more studies were recently completed and reported. The first was conducted at Pharmacia in Italy and examined the effect on micronuclei induction in mouse bone marrow cells by prefolic treatment (PULCI et al. 1995). Groups of 10 male B6C3F 1 mice per group received SASP alone, SASP with prefolic, or prefolic alone (table 1). SASP was given at 1250,2500 and 5000 mg/kg/day (20 ml/kg) for 3 days. Prefolic was given i.v. at a dose of 50 mg/kg/day for 7 days before and during the 3-day treatment with SASP for a total of 10 days. Cyclophosphamide (50 mg/kg i.p.) given once was used as a positive control. The dose of folate given is about 500 times the human dose in methotrexate rescue (WILLIAMS et al. 1990). The micronucleus assay was carried out according to the method reported by MACGREGOR et al. (1987). The slides were stained with Giemsa (GOLLAPUD! and KAMRA 1979). At least 2000 PCEs per mouse were scored. The percentages of micronucleated PCEs, the ratios of PCEs to normochromatophilic erythrocytes (NCEs), and serum folate levels were evaluated and analyzed using Bartlett's homogeneity of variance and Duncan's multiple range analysis tests (DUNCAN 1955). The serum

Table 5: Summary of incidence (%) of all pertinent effects in male mice in the 2-year gavage study with high dose SASpa on three different modes of feed allotment.

0,

Dose (mg/kg) Body Weight changes

= = = = = = = = = = = = = = = =

0,

2700

2700 - groups 12 % lower than control

Nonneoplastic effects Cytol. alter Liver: c.c. focus E. focus Neoplastic effects Liver: H. ad. H. carc. H. ad. & H. carc. Lung: Alv. br. ad. & carc. Stomach: Sqs. c. carc. MO: Lymphoma

Hemosiderin HCP MO Cytol. alter c.c. focus E. focus F.c. hypo H. ad. H. carc. Sqs. c. carc. Alv. br. ad. & carc. **

Weight-Matched Control c

Ad libitum Control b

Mode of Feeding

4 12

22 44

26 26 4 28 6 10

84** 16 12 12 0 2**

Restricted-Feed Control d

0,

2700

2700 - group similar to control

4 2 16 0 0 18 10 9

22

2700 - group 10 % lower than control

0 0

44 84** 0 0 22 0 0**

2700

0 0 0 25 4 8

0 0 0 0 0 6 4 0**

All data were obtained from NTP Technical Reports 457, 1995a and 460, 1995b. Ad libitum control and high (2700 mg/kg/day) dose groups. The daily feed allotment of the control group was restricted so that its mean body weight matched the one of the high dose group. The daily feed offered to both control and high dose groups was approximately 85 % of the ad lib fed control group. hemosiderin pigmentation present. hematopoietic cell proliferation. malignant lymphoma. hepatocellular cytologic alterations. clear cell focus. eosinophilic focus. follicular cell hyperplasia. hepatocellular adenoma. hepatocellular carcinoma. squamous cell carcinoma alveolar/bronchiolar adenoma and carcinoma. significantly different at p < 0.01 from the control group.

folate levels were determined by radio immunoassay (1 25 1), from blood drawn from the aorta 24 hours after the last dose. 32p-Postlabeling Study: The potential of SASP to form DNA adducts in rat (Fischer 344) and mouse (B6C3F) liver and urinary bladder DNA, was assessed by 32P-postlabeling method in a study conducted at the American Health Foundation in New York (IATROPOULOS et al. 1996). SASP (in com oil) was delivered daily by gavage for 7 days at 0, 300, or 1000 mg/kg/day to male rats or at 0, 1000 or 3000 mg/kg/day to male mice. Acetylaminofluorene (2AAF) at 10 mg/kg/day and 2-naphthylamine (NA) at 20 mg/kg/day (both in com oil) were given by gavage for 7 days and were used as positive controls for the liver (2AAF) and urinary bladder (NA). Each group had 4 animals. Drug suspensions were analyzed for dose verification, stability and homogeneity. Serum for achieved drug level was obtained from all SASP-exposed animals , at 4 and 8 hours after the first and last (day 7) doses. Analyses were performed by the methods described above. The livers and urinary bladders were processed for 32P-postJabeling using the PI

enhancement procedure (RANDERATH et al. 1989), which is a very sensitive method capable of detecting single adducts in 109 - 1010 nucleosides from 1-10 Ilg of DNA.

Results Pharmakokinetic Studies Low systemic exposure of SASP was seen in both rat and mouse. The reason is probably a poor absorption, high first pass elimination and an extensive biliary excretion. There was a proportional increase in plasma concentration with dose in the rat. SASP was bound mainly to serum albumin (> 99 %) in both species. Accordingly, the volume of distribution was small. Most of the intravenous dose of SASP was found in the gastrointestinal tract, as a consequence of high biliary excretion. The elimination of radioactivity from tissues followed the elimination from plasma without any appreciable specific activity in any tissue. Exp Toxic Pathol 49 (1997) 1- 2

21

Table 6. Summary of in vitro and in vivo genotoxic effects of SASP, SP or ASA. Type of Assay

Compound

Result (Effect)

Bacterial point mutation a.

Salm. ty.

b.

E. coli

TA95 TA98 TA 100 TA 1535 WP2p, WP2 uvrA-p

with or without S9 with or without S9 with or without S9 with or without S9 with or without S9 with or without S9

SASP, SASP, SASP, SASP, SASP SASP

with or without S9

SASP

SP, ASA SP, ASA SP, ASA SP, ASA

Mammalian point muttions a.

Mouse lymphoma HGPRT

Chromosomal changes a.

b.

c.

CA CA CA SCE SCE SCE CA Human lymphocytes SCE MN SCE MN CA SCE MN Human lymphocytes 1. Crohns patients CA SCE 2. UC patients MN SCE 3. IBD patients MN Chinese hamster ovary cells

with or without S9 with or without S9 with or without S9 with or without S9 without S9 with or without S9 with or without S9 without S9 without S9 with S9 with S9

SASP SP ASA SASP SP ASA SASP, SP SASP SASP SP SP ASA ASA ASA SASP SASP SASP SASP SASP

+

+ + +

(+) + + +

In in vivo chromosomal changes a. b. c.

Mouse, 90 days, peripheral RBCs Mouse, 2 day, bone marrow RBCs Mouse, 13 weeks, peripheral RBCs

MN MN MN

MN/KC CA d. e.

f. g. h. i.

Mouse, 3 days bone marrow RBCs Mouse, 3 days bone marrow RBCs

MN/KC

MN MN Mouse, 3 days, lymphocytes MN CA Rat, 1 day, bone marrow cells Mouse, 3 days, with or without prefolic MN MN Rat and Mouse, 7 days, 32p postlabeling

SASP, SP SASP, SP SASP SASP SASP SASP, SP SP ASA SASP SASP SASP SASP + Prefolic SASP

+ (+) + + + +

+ (+ )/--

CA =chromosome aberrations; SCE =sister chromatid exchanges; MN =micronucleus; MN/KC =kinetochore containing micronuclei; UC = ulcerative colitis; IBD = inflammatory bowel disease; + = positive; - - = negative; (+) = equivocal; (+)1- - = significant reduction

22

Exp Toxic Pathol49 (1997) 1-2

Table 7. Ratio of polychromatic to normochromatic erythrocytes, percentage of micro nucleated polychromatic erythrocytes and serum folate levels in mice: Mean ± SD of values for each experimental group. Group No.

Treatment

Dose (mg/kg/day)

Vehicle

Polychrom.l Normochrom. Erythrocytes Mean ± SD

% Micronucl. Polychrom. Erythrocytes Mean ± SD

0.93 ± 0.17

0.04 ± 0.08

90.0±17.4

Serum Folate levels (mg/mL) Mean ± SD

2 3 4

SASpa SASP SASP

1250 2500 5000

0.89 ± 0.11 0.89 ± 0.22 1.02 ± 0.15

0.28 ± 0.17++ 0.31 ± 0.17++ 0.42 ± 0.18++

56.6 ± 7.1++ 48.2 ± 8.9++ 58.4 ± 10.3+

5 6 7

SASP + Prefolicb SASP + Prefolic SASP + Prefolic

1250 + 50 2500 + 50 5000 + 50

1.07 ± 0.29 1.02 ± 0.27 1.07 ± 0.28

0.12 ± 0.11 . O.l7±0.12 0.24 ± 0.11 +.

107.7 ± 13.1 .. 116.4 ± 15.0+" 117.7±41.1+"

8

Prefolicc

50

0.83 ± 0.17

0.08 ± 0.05

155.0 ± 37.0++

9

Cyclophosphamided

50

0.91 ±0.18

1.57 ± 0.33**

nd

a = administered by gavage for 3 days. b = administered intravenously 7 days before and for 3 days during SASP treatment. = administered intravenously for 10 days. d = administered once intraperitoneally. nd = not determined. ** = p < 0.001 comparing cyclophosphamide and vehicle control (Mann-Whitney U-test). + = p < 0.05; ++ = p < 0.01 compared to vehicle control (Duncan's multiple range test). = p < 0.05; •• = P < 0.01 compared to SASP alone at corresponding doses (Duncan's multiple range test).

The major part of both oral and intravenous dose was excreted in the feces (> 90 %). The principal metabolism of SASP consisted of azocleavage to ASA and SP in the lower intestine by microflora. Both metabolites, especially SP, were found in plasma at higher concentrations than SASP. Further metabolism included hydroxylation and conjugation with acetyl, glucuronide and sulfate for which male rats showed highest extent.

Genotoxicity Studies In the bacterial mutation tests, SASP at 0.4 to 6250 I1g/plate did not display mutagenicity in the Salmonella typhimurium tester strains T A98 and TAl 00 or E. coli WP2p and WP2 uvA-p, with or without S9 fraction (table 6). Likewise, in the mouse lymphoma gene mutation assay, SASP was negative (table 6). SASP also did not cause CAs in lymphocytes or in vivo in rat bone marrow cells (table 6). Cultured human lymphocytes from ulcerative colitis patients treated with SASP gave negative MN and positive SCE results. Lymphocytes from Crohn's disease patients were weakly positive, while lymphocytes from patients with inflammatory bowel disease gave positive SCE and MN results (table 6).

Folate Supplementation Study The summary of data from the study on the effect on MN induction in mouse bone marrow cells by prefolic treatment is given in figure 1 and table 7. No significant differences were found in the ratio of PCEs to NCEs of any treatment groups. This indicates that none of the SASP or prefolic treatments were cytotoxic to bone marrow cells. SASP, at all 3 dose levels given daily for 3 days, significantly increased (p < 0.01) the frequency of micronucleated PCEs over the vehicle control group. These increases were accompanied by a significant decrease in serum folate levels (p < 0.05) at all tested doses. Treatment with prefolic daily (at 50 mg/kg/day) for 10 days, resulted in a 72 % increase in serum folate levels over the control (p < 0.01), without increasing the frequency of MN. The serum folate levels in mice given prefolic before and during SASP treatment were about 20 to 31 % higher than those of the control group. The increase was associated with a reduction in the frequency of MN compared to the groups on SASP without prefolic. The increases in serum folate levels were able to antagonize the SASP effect, since the frequency of MN was not different from that of the control, except at the high dose of SASP, at which the frequency was still significantly higher (p < 0.05). Exp Toxic Pathol49 (1997) 1-2

23

045 040 035 030

t025 ~ 020

z

~ 015

010

005 O ~~~~rL~rL~rL~~

+F

+F

C = Control; S = SASP; F = Folic; MNPE = Mononucleated polychromatic erythrocytes. Fig. 1. SASP and MNPE. * P < 0.05, ** P < 0.01 compared to control. + P < 0.05, ++ P < 0.01 compared to same dose of SASP

32P-Postlabeling Study In the DNA adduct study, the serum SP levels in rats documented a dose-dependent exposure to SASP throughout the study, whereas in mice serum SP levels plateaued at 3000 mg/kg after the seventh dose. In the SASP autoradiograms of liver DNA from both species, no modified base (adduct spots) was evident. The positive control, 2-AAF, revealed adduct spots in the DNA from livers of both species. Also, some I-compounds (naturally occurring indigenous modified bases) were present in both SASP-exposed and untreated rats and mice. In the urinary bladders of both species, there was likewise no DNA adduct spot in any of the autoradiograms of SASPtreated rats and mice. NA, the positive control, produced modified bases in rat urinary bladder DNA.

Discussion SASP was reported to produce increases in rat urinary bladder and mouse liver tumors under ad libitum feeding conditions (NTP 1995a; 1995b; ABDO and KARl 1996). When feed restriction regimens were used, these tumors were absent in both species (ABDO and KARl 1996). The present investigations provide further insight into the implications of these findings for the safety of SASP in humans. In all species studied including man, orally administered SASP undergoes azoreductive cleavage by the colonic bacterial flora and is bioconverted into 5-aminosalicylic acid and sulfapyridine (NTP 1994). In rats, mice and man, SASP is poorly absorbed from the intestines. Rats absorb SASP more poorly than mice. From the products of colonic cleavage, SP is well absorbed and is excreted in the urine. ASA is poorly absorbed and is excreted in the feces. As a rule, in the intestine, with 24

Exp Toxic Pathol49 (1997) 1-2

increasing SASP dose there is a decrease in absorption efficiency (PEPPERCORN and GOLDMAN 1972; CHUNGI et al. 1989; TETT 1993; ZHENG et al. 1993; NTP 1994). Unlike man, rats and mice clear the parent compound to a high extent in the bile. SP is eliminated more slowly in male rats compared to male mice. In the present investigations in the 32P-postlabeling study, serum SP levels revealed that both rats and mice were exposed to high levels of SASP. In the rat there was dose proportionality at all sampling times, with a decrease in clearance by day 7, whereas in the mouse, absorption was saturated at 3000 mg/kg of SASP, after the seventh dose. In the liver, SASP does not undergo further reduction, but instead is hydroxylated and conjugated to acetyl, glucuronides and sulphate metabolites (ZHENG et al. 1993). Conjugation is, in general, more extensive in males (PREISIG 1983). The mouse clears SASP more readily through the bile than either rat or man (ZHENG et al. 1993), and on return to the intestine, SASP can be azoreduced to SP, which is readily absorbed and excreted via the kidney, along with the small amounts of SASP, which escaped hepatic clearance. Since the ratio of liver volume and plasma flow rate in the rat is 2.4 times lower and 1.3 times higher in the mouse compared to man (IATROPOULOS 1993; IATROPOULOS et al. 1994a), hepatic clearance of both parent compound and metabolites is lower in the rat and higher in the mouse compared to man. Neither SASP nor any of its metabolites have been reported to produce any gene mutational activity in any in vitro tests (ZEIGER et al. 1990). SP and SASP do induce increases in SCEs and MN in in vitro cultured lymphocytes (MACKAY et al. 1988; BISHOP et al. 1990) in the absence of S9 (table 9). SP alone causes increases in SCE in CHO cells in the absence of S9. Similar findings were confirmed in various in vivo mouse studies, i.e. increases in MN of peripheral and bone marrow RBC by SASP and SP. No CA were present in any of the mouse or rat assays (BISHOP et al. 1990), and only equivocal findings were made in man (MITELMAN et al. 1982). These chromosomal changes do not reflect clastogenicity; rather they indicate the presence of aneuploidy (GUDI et al. 1990) caused by a failure of mitotic chromosomal segregation. This was confirmed in the micronucleus/kinetochore staining assay (WITT et al. 1992a; WITT et al. 1992b). The level of SCE increases are similar in both in vitro (higher increases) and in vivo assays, although exposure to cytostatic drugs in vivo (attending nurses) have shown that SCE have a relatively short lifespan and they are reversible (NORPPA et al. 1980). Increases in MN of bone marrow cells were reported in both male and female mice exposed to SASP (NTP 1995a). No evidence of any structural CA in bone marrow cells of male mice was found, confirming previous findings. When male rats were used at equitoxic doses, no increases in the frequency of MN in bone marrow cells was reported (NTP 1995a). These findings thus indicate a species difference. They also confirm the lack of

clastogenicity and demonstrate that the mouse RBC have a distinct sensitivity to SASP. This is further confirmed in a 13-week mouse study, where an anemia was present in both genders, probably related to methemoglobinemia (NTP 1995a). From all genotoxicity assays, it is evident that SASP and all of its major metabolites do not exhibit any mutagenic activity and do not adduct DNA. On the other hand, SP and SASP have induced SCE and MN in cultured human lymphocytes, without the presence of liver activation enzymes. They can also induce MN in mice bone marrow and peripheral RBC. This effect is not present in the rat. In the present study, treatment with prefolic daily for 10 days, increased serum folate levels, while reducing the frequency of MN induced by SASP in a dose-related pattern in mice (fig. 1, table 7). Thus, serum folate levels are causally related to MN induction. SASP impairs folate absorption from the intestine (FRANKLIN and ROSENBERG 1973; SELHUB et al. 1978) and folate is a cofactor for DNA synthesis. Low folate leads to utilization of uridine in DNA in place of thymidine, which in tum leads to DNA strand breaks in vivo (JAMES and YIN 1989), CA (LI et al. 1986), MN formation in vivo and in vitro (JACKY et al. 1983) and to aneuploidy (WITT et al. 1992b), which reflects failure of mitotic chromosomal segregation. When folate was supplemented, MN were significantly reduced. Failure to completely block the SASP-induced increase in MN may be due to other SASP / folate interactions not reflected in serum folate levels. Perhaps the differences between mice and rats can be explained by differences in RBC lifespan (mouse 28 and rat 58 days) (JAIN 1986), as well as sustained blood levels of SP, as was also documented in the 32P-postlabeling study, where achieved SASP serum levels were higher in the mouse. In a 13-week mouse study (NTP 1995a; 1995b), a significant reduction in body weight occurred in the high dose groups of both genders. The liver weights of both genders of all SASP-exposed groups were increased. Microscopically, 50 % of the mid and 100 % of the high dose males showed centrilobular hepatocellular hypertrophy. By week 103-104 (in the 2-year study), there were increases in the incidence of eosinophilic foci of hepatocellular alteration (HAF) and a significant reduction in follicular cell hyperplasia of the thyroid in all SASP-treated groups of both genders (table 4). The effects on the thyroid may be related, in part, to the reduced body weight gain caused by SASP. Furthermore, in both genders, hepatocellular neoplasms were increased comparably in all SASP-exposed groups from 48 % to 88 % and a very significant decrease occured in multiorgan malignant lymphoma from 10 % to 2 %. In the WM and RF high dose groups, no hepatocellular carcinomas (ABDO and KARl 1996), or in the case of RF high dose group, no hepatocellular adenomas were evident (table 5). Also, the multiorgan malignant lymphoma reduction in the high dose ad libitum (AL) groups was evident in both WM and RF high dose groups (ABDO and KARl 1996). The reason for the decreased incidence of lymphoma in SASP-trea-

ted mice is not entirely clear. However, similar results have been obtained with other compounds evaluated by NTP. Additional studies on this potentially important finding appear to be warranted. The influence and modulation by food intake on development of mouse liver tumors is well established (TUCKER 1985; LAGOPOULOS and STALDER 1987). Consequently, in this species, the liver neoplasia is not directly attributable to SASP, whereas the decrease in incidence of lymphoma is associated with SASP treatment. In a similar I3-week rat study as in the rat (NTP I995a; 1995b), males of the mid and high dose group and females of the high dose group, showed increases in kidney weights, a minimal follicular cell hyperplasia in the thyroids, enlargement of the TSH-producing cells in the pituitary, increase in urinary volume and a decrease in urine specific gravity. In the mid and high dose males, there were also decreases in T3 and T4 , increases in TSH serum concentrations, and lower sperm motility. In the subsequent 2-year study, by week 60 in both genders, there was continuation of the renal effects, especially increase in urinary pH, as well as a splenic extramedullary erythropoiesis (table 2). By week 103-104, hyperplasia of the urinary bladder and renal pelvis was present in the top two dose groups of both genders and the occurrence of urinary bladder papillomas in the mid (4 %) and high (12 %) dose males and mid (4 %) dose females. In addition, in high dose females there were also renal papillomas (4 %). There was a significant reduction in MCL in multiple organs in the high dose groups of both genders. In the WM high dose group, the reduction was similar to the AL group (table 3). In the RF high dose group, no papillomas were present although the pattern ofMCL was still significantly reduced (ABDO and KARl 1996). These two findings, that is, a dietary effect on papillomas but not on MCL, indicate that the increase in papillomas is not directly related to SASP, whereas the reduction in leukemia is associated with SASP. In considering the pathogenesis of both nonneoplastic and neoplastic SASP-associated changes in the rat, certain physiological gender differences become evident. For example, the urinary pH in females is higher, while the urine specific gravity is lower compared to that in males (IATROPOULOS et al. 1994b). Both of these parameters were affected, i.e. pH was increased and specific gravity was reduced, in the high dose SASP groups of the chronic rat bioassay, measured during the 60 week interval. At the 103-104 week interval, the urothelial hyperplasia in both kidney and urinary bladder, and the papillomas, in the males primarily, followed the same trend. When feed was restricted, hyperplasia in the kidneys of high dose SASP groups was reduced from 86 % in both AL and WM, to 36 % in RF group. Likewise, the urinary bladder hyperplasia diminished from 82 % (in AL and WM rats) to 14 %, and the calculus formation from 54 % to O. Unfortunately, no urinalysis data are available for the 104 week segment of study. At the same time, the incidence of multi organ leukemia was reduced Exp Toxic Pathol 49 (1997) 1-2

25

in all 3 high-dose SASP groups, e.g. AL, WM, and FR. This last effect is in agreement with SASP antiinflamatory activity and efficacy (CARLIN et al. 1985; CARLIN et al. 1989; REMVIG and ANDERSEN 1990). Furthermore, decreasing food consumption reduces the average daily body temperature, the whole animal Oz consumption, drug metabolizing enzymes, rate of water consumption, which in tum alters corticosterone, insulin and thyroid hormone homeostases (HART et al. 1995; 1996), as well as causing oxidative DNA damage (DJURIC et al. 1992). In the long term rat studies, the factors mentioned above have also modified the luminal conditions of urothelial cell lined cavities, to result in sustained pH increases and specific gravity reductions, which alone are capable of inducing chronic urothelial stimulation, concretions, urothelial hyperplasia and neoplasia (lATROPOULOS et al. 1994b). These luminal conditions have not been reported in patients receiving SASP and are specific to the conditions of exposure and response in the rat. In the mouse, in both genders, there was a significant reduction in body weight in high dose SASP animals. Liver weights were also increased in SASP-exposed animals, with centrilobular hepatocellular hypertrophy at first (at 13 weeks) and then later (at 104 weeks) increases in the incidence of hepatocellular altered foci. As per known pharmacokinetic data, after SASP, SP and ASA and other minor metabolites enter the liver, there is no further reduction. Instead, there is hydroxylation, conjugation and excretion in the bile, which occurs more readily in males than in females, and in mice more readily than in either rat or man, because of species differences in hepatic clearances (lATROPOULOS 1993; IA TROPOULOS et al. 1994a). In addition, inhibition of weight gain over time, causes modulation of intermediary metabolism, with shifts away from proper fat utilization (MATHEWS and BATEZZATI 1994). Furthermore, a reduction in folate absorption caused by SASP results in depletion of liver folate stores and interferes with DNA synthesis, causing impairment of normal cell division throughout the body, but especially in rapidly proliferating RBC precursors (RBC lifespan 28 days in mice, 58 days in rats and 130 in man). This was manifested in anemia, methemoglobinemia and induction of MN in mice only. It has also been reported that chronic folate deficiency results in liver DNA hypomethylation, which renders genomic and p53 DNA prone to breaks, chromatin alterations and sensitive to oxidant-induced DNA injury and subsequent neoplasia (POGRIBNY et al. 1995). Thus, these chronic events lead to hepatocellular overburden, resulting in degeneration, genomic and p53 DNA injury, necrosis and subsequent proliferation, and neoplasia. Accordingly, the mechanism of hepatocellular neoplasia is not a direct effect of SASP, but a combination of indirect effects, which are species (mouse) specific and not relevant to man. On the other hand, the reduction in the incidence of the multiorgan lymphoma was consistently evident in all SASP high dose groups, i.e. AL, WM and RF, because it is part of SASPs spectrum of activity. From the 3zP-postlabeling 26

Exp Toxic Pathol49 (1997) 1-2

study, it is evident that SASP, at the same high doses used in the 2 bioassays, was not capable of forming DNA adducts in liver or urinary bladder DNA of rats and mice (IATROPOULOS et al. 1996), which also argues against a direct carcinogenic effect. To summarize, neither SASP nor its major metabolites, is genotoxic or capable of directly inducing neoplasia. The occurrence of liver neoplasms in the mouse and urinary bladder papillomas in the rat are the result of species specific effects which have not been encountered in patients receiving SASP therapeutically. On the other hand, SASP is associated with reduction of hematolymphopoietic neoplasia in both rats and mice. Also, ulcerative colitis patients on SASP show a low incidence of colorectal cancer (ODES and FRAZER 1989), and upon receiving folate supplementation, show reduced incidences of neoplasia (LASHNER et al. 1989). Finally, several epidemiological studies have been conducted, taking advantage of the long use of SASP during the last 50 years. The results demonstrate no association between SASP use and increased risk of neoplasia at any site in man (EKBOM et al. 1991; PINCZOWSKI et al. 1994). Accordingly, we conclude that SASP does not pose a cancer risk to humans at the therapeutic dosages.

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