An adverse outcome pathway for small intestinal tumors in mice involving chronic cytotoxicity and regenerative hyperplasia: a case study with hexavalent chromium, captan, and folpet

Figures & data

Figure 1. Proposed AOP for cytotoxicity-mediated SI cancer in mice. (A) AOP diagram. See text for discussion regarding (M)IE. Future evolution of this AOP may better define the (M)IE associated with villous cytotoxicity. (B) Structure of intestinal mucosa. Villi are finger- or leaf-like projections into the lumen that are predominantly covered with mature, absorptive enterocytes, along with occasional mucus-secreting goblet cells. These cells live only for a few days, then die and slough into the lumen. The crypts, or glands of Lieberkühn, are tubular invaginations of the epithelium, lined largely with younger epithelial cells, which serve as a source of enterocytes to multiple villi. At the base of the crypts are stem cells, which divide continually and function as the source of all the epithelial cells in the crypts and on the villi. Adapted from O'Brien et al. (2013).

Figure 2. Synchrotron-based XRF microscopy of Cr in Swiss roll preparations of a duodenum from a mouse exposed to 180 ppm Cr(VI) for 7 days. (A) XRF for calcium (Ca) indicates presence throughout the intestinal mucosa. (B) XRF for chromium (Cr) indicates presence in villi but not crypts (white dotted circles). Adapted from Thompson, Wolf, et al. (2015).

Table 1. Incidence of Cr(VI)-induced crypt hyperplasia in mice.

ppm Cr(VI) in drinking water	Time (Days)
	7	28*	28 + 28*	90†	90‡	728
0	0/5	0/10	5/10	0/10	0/20	0/81
0.1	0/5	–	–	0/10	–	–
1.4	0/5	–	–	0/10	–	–
5	0/5	–	–	0/10	–	27/85
10	–	–	–	–	–	18/45
20	0/5	–	–	0/10	11/20	35/48
30 LOAELtumor	–	–	–	–	–	42/48
40	–	–		–	13/20	–
60	3/5	–	–	9/10	–	31/42
90	–	–	–	–	20/20	32/40
180	5/5	10/10	5/10	9/10	20/20	42/48
350	–	–	–	–	20/20	–

* Thompson, Wolf, et al. (2017): 28-day exposure followed by 28-day recovery period (see text).

† Thompson, Proctor, et al. (2011).

‡ NTP (2008).

Table 2. Incidence of folpet- and captan-induced crypt hyperplasia in mice*.

Folpet in diet (ppm)	Time (Days)
	1	3	7	14	28	28 + 17†	28 + 28†	2 years
0	0/5	0/5	0/5 0/12	0/5 1/12	0/12	0/12	5/10	–‡
1000	–	–	–	–	–	–	–	NOAEL hyperplasia‡
3000	0/5	4/5	5/5	5/5	–	–	–
5000 LOAELtumor	0/15	–	–	–	–	–	–	M + F‡
6000	–	–	12/12	12/12	23/24 9/10	9/24	7/10	M + F‡
12 000	–	–	–	–	–	–	–	M + F‡
16 000	–	–	–	–	10/10	–	4/10	–
Captan in diet (ppm)	Time (Days)
	1	3	7	14	28	28 + 28†	56	90	560	2 years
0	0/5	0/5	0/5	0/5	–	5/10		–¶	–¶	–§
400	–	–	0/5	–	–	–	NOAEL hyperplasia	–	–	–
800	–	–	–	–	–	–	F	–	–	–
3000	0/5	4/5	5/5	5/5	5/5	–	M + F	–	–	–
6000 LOAELtumor	–	–	–	–	10/10 M¶	6/10	M + F M¶	M¶	–	M + F§
16 000/12 000	–	–	–	–	9/10	2/10	–	–		–
16 000	–	–	–	–	–	–	–	–	M + F¶	M + F§

M = male F = female.

* Compiled from FSCJ (2017), Gordon et al. (2012), JMPR (1996, 2004a, 2004b), Thompson, Proctor, et al. (2011), Thompson, Wolf, et al. (2017), Wilkinson et al. (2004), and U.S. EPA (1995).

† Exposure followed by recovery period.

‡ FSCJ (2017)- incidence not reported, lowest tested concentration.

¶ Wilkinson et al. (2004)- incidence not reported (females not examined).

§ U.S. EPA (1995)- incidence not reported.

Figure 3. Qualitative and quantitative evidence for sustained crypt hyperplasia following exposure to Cr(VI). Full transverse sections indicate the extent of villous blunting in mice exposed to 180 ppm Cr(VI) for 90 days (B) relative to controls (A). Higher magnification demonstrates the extent of crypt elongation in exposed mice (D) relative to controls (C). The arrows demonstrate blunting of villi, and the brackets indicate crypt expansion. (E) Cells per crypt and the incidence of crypt hyperplasia in mice exposed to various concentrations of Cr(VI) for 7 days. (F) Cells per crypt and the incidence of crypt hyperplasia in mice exposed to various concentrations of Cr(VI) for 90 days. Adapted from Thompson, Seiter, et al. (2015); Thompson, Wolf, et al. (2015); Thompson, Suh, et al. (2017).

Figure 4. Qualitative evidence for sustained crypt hyperplasia following exposure to captan for 28 days. (A) Representative transverse section of control mouse SI. (B) Representative transverse section of SI in a mouse exposed to 6000 ppm folpet in diet. Folpet-induced short term cytotoxic and proliferative changes in the mouse duodenum, Gordon et al., Toxicology Mechanisms and Methods, reprinted by permission of Informa UK Limited, trading as Taylor & Francis Group, www.tandfonline.com

Table 3. Summary of in vivo duodenal genotoxicity assays for Cr(VI), captan, and folpet.

Compound	Micronucleus (duodenum)	Mutation (duodenum)	Supporting data (duodenum)
Cr(VI)	O’Brien et al. (2013): no crypt MN in duodenum after 90 days of exposure to ≤180 ppm Cr(VI). Thompson, Wolf, et al. (2015): no increase in crypt MN in duodenum after 7 days of exposure to ≤180 ppm Cr(VI).	O’Brien et al. (2013): no change in kras codon 12 GGT->GAT MF in duodenum after 90 days of exposure to ≤180 ppm Cr(VI). Thompson, Young, et al. (2017): no increases in cII MF in TGRF344 rats exposed to 180 ppm Cr(VI) for 28 days. Aoki et al. (2019): no increases in gpt MF in gpt delta mice exposed to 30 or 90 ppm Cr(VI) for 28 days; no increase in gpt MF in mice exposed to 3–30 ppm Cr(VI) for 90 days.	De Flora et al. (2008): no increases in DPC or 8-OHdG were observed in the duodenum of SKH-1 mice exposed to 20 ppm Cr(VI) for 9 months. O’Brien et al. (2013): dose-dependent increases in Cr-DNA binding were measured and reported as Supplemental material. However, analytical evidence of ex vivo binding in the assay confounds the Cr-DNA binding observation. Thompson, Seiter, et al. (2015) and Thompson, Wolf, et al. (2015): synchrotron XRF microscopy demonstrated localization to intestinal villi but not crypts. Findings support negative genotoxicity results in crypts and indicate that any Cr-DNA binding occurred in villous enterocytes. Thompson, Seiter, et al. (2015): λ-H2AX immunostaining was evident in in villi villous tips but not in crypts. Thompson, Seiter, et al. (2015); Thompson, Wolf, et al. (2015): No aberrant crypt-like foci in villi (visible by H&E stain) were identified in mice exposed to ≤180 ppm Cr(VI) for 13 weeks.
Captan	Chidiac and Goldberg (1987): no increase in crypt MN in duodenum after 1 week of exposure to ≤4000 mg/kg captan in diet.	FSCJ (2017): mentions unpublished results indicating negative results “obtained from a gene mutation assay of the target in transgenic mice… Therefore, a genotoxic mechanism was unlikely involved in the tumor development, and it enabled us to establish a threshold in the assessment.”	Arce et al. (2010): summary of radiolabeled captan studies indicates no captan-DNA binding in the duodenum.
Folpet	Gudi and Krsmanovic (2001) (as described in Arce et al. (2010): no increase in crypt MN in duodenum after 1 week of exposure to 2000 mg/kg folpet in diet.		Clay (2004) (as described in Arce et al. 2010): no increase in DNA damage in duodenal crypts (via Comet assay) was observed 2 or 6 hr after exposure to 2000 mg/kg folpet

Table 4. Support for essentiality of key events (KEs).

	Defining question	High (strong)	Moderate	Low (weak)
	Are downstream KEs and/or the AO prevented if an upstream KE is blocked?	Direct evidence that downstream KEs and/or the AO prevented if an upstream KE is blocked?	Indirect evidence that downstream KEs and/or the AO prevented if an upstream KE is blocked?	No or contradictory experimental evidence of the essentiality of any of the KEs.
(M)IE: villous enterocyte cytotoxicity	STRONG. There is direct evidence of the localized absorption of Cr to the villus (but not crypt) compartment as early as 7 days after oral exposures to Cr(VI) at 180 ppm with localization remaining in the villi (and not crypt) at 13 weeks. There is indirect evidence via radiolabel studies for the duodenal localization of captan and/or its metabolites. Villous enterocyte cytotoxicity is observed as early as after 1–7 days after Cr(VI), captan, or folpet exposure at concentrations equivalent to or lower than tumorigenic concentrations. An initial molecular target(s) for Cr(VI) and captan/folpet may not be required or exist. Pharmacological depletion of glutathione (GSH) can induce cytotoxicity by shortening intestinal villi and Cr(VI) and captan/folpet are known to affect cellular GSH and redox status. The cytotoxicity evident after 28 days is reversible after a 28-day recovery period, providing direct evidence of essentiality.
KE1: sustained crypt cell proliferation/ hyperplasia	STRONG. Long-term exposure plus reversibility (≥6-month) studies with captan show that cessation of exposure to carcinogenic doses prevents or lowers the tumor incidence suggesting that crypt hyperplasia must be sufficiently sustained across dose and time and that sufficient daily exposure durations may exceed 6 months to result in small intestinal tumors. For Cr(VI), the duodenal tumors had a long latency with the first incidence of adenoma at 451 days (∼15 months) in males or 693 days (∼23 months) in females and the first incidence of carcinoma at 729 days (terminal sacrifice, 18 months) in males or 625 days (20 months) in females (NTP 2008).
KE2: mutation/transformation	STRONG. Carcinogenesis requires acquisition of multiple mutations resulting in phenotypic transformation from a stem cell to an initiated cell capable of tumor formation. In Cr(VI) or captan/folpet studies, no SI tumors were observed at concentrations not also inducing sustained crypt hyperplasia. The absence of induced mutations in short- and intermediate-term target tissue assays (up to 13 weeks) for Cr(VI) and captan is strong evidence that spontaneous mutation/transformation occurred later in the carcinogenic process secondary to sustained cytotoxicity and regenerative cell proliferation.

Table 5. Support for biological plausibility of key event relationships (KERs).

	Defining question	High (strong)	Moderate	Low (weak)
	a) Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?	Extensive understanding of the KER based on extensive previous documentation and broad acceptance.	KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete	Empirical support for association between KEs, but the structural or functional relationship between them is not understood.
(M)IE = > KE1: villous enterocyte cytotoxicity leads to sustained crypt cell proliferation/hyperplasia	STRONG. The sustained localization of a stressor to the duodenal villi (but not crypt), even after 13 weeks of continuous exposure based on direct evidence with Cr(VI), is plausibly attributed to the efficiency of the intestinal epithelial barrier, which functions to prevent the transfer of harmful agents from the gut lumen into the circulation via the subjacent tissue (i.e. crypt), lymphatics, and vasculature. While evidence could be considered Moderate because sustained localization has only been directly demonstrated for Cr(VI), the sequalae for captan and folpet, which includes interepithelial lymphocytes, could be considered STRONG evidence because it closely mimics celiac disease (thereby increasing biological plausibility). It is broadly accepted that repeated stressor-induced cytotoxicity at the site of target tissue contact is associated with regenerative cell proliferation in response to repeated injury. It is broadly accepted that sustained exposure to a cytotoxic chemical increases regenerative cell proliferation and leads to tissue hyperplasia. In the mouse small intestine, primarily in the duodenum, crypt hyperplasia is observed in every strain of tested mice, specifically B6C3F1, BALB/c, and AM3-C57BL/6 mice exposed to Cr(VI), B6C3F1 mice exposed to captan, and B6C3F1 and CD-1 mice exposed to folpet.
KE1 = > KE2: sustained crypt cell proliferation/hyperplasia leads to mutation and transformation	STRONG. It is broadly accepted that increased cell proliferation increases the chance for spontaneous mutations to arise leading to cell transformation leading to neoplasia. The duodenum is an ideal target organ for examining genotoxicity, especially when it is the cite of carcinogenic action. The absence of positive genotoxicity results in the duodenum of multiple studies strongly supports that genotoxicity is not an early KE and therefore must occur as a later KE in the etiology of chemical-induced SI tumor formation.
KE2 = > AO: mutations and transformation lead to small intestinal tumors in mice	STRONG. Numerous studies with multiple chemical stressors demonstrated that sustained crypt hyperplasia in mice precedes duodenal tumors in mice. There is no inconsistent evidence.

Table 6. Empirical support for key event relationships (KERs).

	Defining question	High (strong)	Moderate	Low (weak)
	Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown? Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup > than that for KEdown? Inconsistencies?	Multiple studies showing dependent change in both events following exposure to a wide range of specific stressors. No or few critical data gaps or conflicting data	Demonstrated dependent change in both events following exposure to a small number of stressors. Some inconsistencies with expected pattern that can be explained by various factors.	Limited or no studies reporting dependent change in both events following exposure to a specific stressor; and/or significant inconsistencies in empirical support across taxa and species that do not align with hypothesized AOP
(M)IE = > KE1: villous enterocyte cytotoxicity leads to sustained crypt cell proliferation/ hyperplasia	STRONG. At exposure concentrations associated with downstream KEs and the AO, intense Cr localization to duodenal villi (but not crypt) after 1- or 13-week exposures has been demonstrated using synchrotron-based x-ray fluorescence microscopy. Radiolabel derived from captan and/or its metabolites has also been measured in mouse duodenal contents (but not epithelium) after dietary captan exposures associated with downstream KEs and the AO. There is no evidence of villous enterocyte cytotoxicity occurring without the duodenal localization of chemical stressor and/or its metabolites. Crypt cell proliferation/hyperplasia is considered a reparative or regenerative process in response to the loss of enterocytes from the villi. There is direct molecular, cellular, and histological evidence of crypt cell hyperplasia in response to chemical exposure at tumorigenic concentrations in a dose and temporal fashion after as short as 1 to 7 days. There is no evidence of regenerative cell proliferation or sustained crypt hyperplasia occurring without concurrent villous enterocyte cytotoxicity in dose or time. There are no data gaps or inconsistencies.
KE1 = > KE2: sustained crypt cell proliferation/ hyperplasia leads to mutation and transformation	STRONG. It is broadly accepted that increased cell proliferation/hyperplasia provides more opportunities for spontaneous mutations to arise leading to cell transformation and ultimately neoplasia. The WOE within and among each of the proposed chemical stressors provides consistent and direct evidence that crypt hyperplasia must be sufficiently sustained across dose and time to result in tumors. Recovery studies with Cr(VI), captan, and/or folpet show that the cytotoxicity-mediated regenerative hyperplasia is reversible for exposures ≤6 months. Further, it is rare that a tumor target site is also an ideal tissue to examine genotoxicity, by being a portal of entry and highly proliferative tissue. Cr(VI) or captan did not induce duodenal mutations or micronuclei after up to 13 weeks of exposure to tumorigenic concentrations. Negative genotoxicity uniquely coupled to corroborative test article imaging for Cr(VI) at the target site is strong support for direct genotoxicity not occurring in the duodenum.
KE2 = > AO: mutations and transformation lead to small intestinal tumors in mice	STRONG. Long-term reversibility studies with captan suggest that sufficient daily exposure durations may exceed 6 or 12 months, due to the high capacity of the small intestine for regeneration and repair, to result in small intestinal tumors. Tumors in Cr(VI)-exposed mice had a long latency (≥15 months) and did not affect survival. While confidence in this KER is considered STRONG, additional studies characterizing interspecies differences in toxicokinetics and toxicodynamics could aid in assessing the human relevance of the AO for regulatory risk assessment purposes.

Table 7. Evolved Bradford Hill causal considerations, defining questions, and body of evidence per OECD (2018).

Evolved Bradford Hill causal considerations (assigned weight)	Defining questions	(M)IE: villous enterocyte cytotoxicity	KE1: sustained crypt cell proliferation/hyperplasia	KE2: mutation/transformation	AO: small Intestinal Tumors
Biological plausibility (weight: NA, qualitative narrative)	Does the hypothesized MOA conflict with broader biological knowledge? How well established is the MOA?	Supporting evidence (strong): the general MOA underlying each of the KEs and KERs is broadly accepted based on well-established scientific knowledge and principles. Unique to this AOP is the (pre)MIEs and the AO. Recognizing the rarity of small intestinal tumors in rodent carcinogenicity bioassays, the AOP and its associated KEs and KERs are considered highly plausible based on the WOE for and against non-genotoxic initiating and KEs. Inconsistent evidence (weak): evidence for a genotoxic MOA is considered weak since positive responses are obtained either in vitro or in non-target tissues after high and/or non-physiological routes of exposure. Genotoxicity evaluations using exposure conditions consistent with the measured KEs and AO, including in the target tissue, are negative.
Quantitative score: strong evidence (+3), moderate evidence (+2), weak evidence (+1) No evidence (0), weak counter evidence (–1), moderate counter evidence (–2), strong counter evidence (–3)
Essentiality of key events (KEs) (weight: 0.4)	Is the sequence of KEs reversible if dosing is stopped or a KE is prevented?	+2*	+3	+3	+3
Empirical support (weight: 0.2 each)	Dose concordance	+3	+3	+3	+3
	Temporal concordance	+3	+3	+3	+3
Consistency (among different biological contexts) (weight: 0.1)	What is the pattern of observations across species/strains/organs/test systems? What would be expected based on the MOA?	+2^†	+3	+3	+3
Analogy (consistency across structurally related chemicals) (weight: 0.1)	Would the MOA be anticipated based on broader chemical knowledge?	+2†	+3	+3	+3
KE weighted sum		2.4	3	3	3
Quantitative confidence score = $\sum$ KE (quantitative WOE score × weight)/possible points = 11.4/12 (95%)

* A specific molecular target for Cr(VI) and captan/folpet may not be required or exist. No pharmacological modulation studies, such as with GSH, exist to demonstrate that blocking the (M)IE prevents downstream KEs.

† While captan and folpet share a common major metabolite, there is no clear structural relationship between Cr(VI) and captan/folpet

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