The Evolving Role for Zinc and Zinc Transporters in Cadmium Tolerance and Urothelial Cancer

Round 1

Reviewer 1 Report

Dear Authors, 

The review you submitted is very interesting and logically constructed. However, even if the English style is fine, some sentences are not very fluent, especially in the introduction section.

Moreover, I have some minor concern in order to improve the quality of your paper.

1- "Aza-dC" is written as "AzadC" sometimes (e.g. Figure 1, panel D). Please fix it.

2- Since Zinc is involved in redox pathways, it should be fine to mention the SOD1 protein (Cu-Zn superoxide dismutase).

3- Again, concerning the SOD1 protein, it should be fine to mention a recent article concerning the role of Cd in altering the structure and function of SOD1 in human cells (doi: 10.1016/j.redox.2019.101102).

4- Focusing on UROtsa cells and their clones (UTCd1-d7), Figure 2 shows only differences between UTCd1 and d6. It should be fine to illustrate all the other clones or, at least, to mention their histogram lack in the main text.

Author Response

Reviewer 1  

We thank the Reviewer for his/her evaluation of our review article and for comments to improve our paper.  We have provided below point-by-point responses to the issues raised.

Comments and Suggestions for Authors

Point 1: The review you submitted is very interesting and logically constructed. However, even if the English style is fine, some sentences are not very fluent, especially in the introduction section.

Response: We have rewritten the first paragraph of the Introduction to state sources of Cd exposure (lines 33-38). An aim of our review article has also been rephrased for clarity (lines 52-53).

Moreover, I have some minor concern in order to improve the quality of your paper.

Point 2: "Aza-dC" is written as "AzadC" sometimes (e.g. Figure 1, panel D). Please fix it.

Response: A correction has been undertaken.

Point 3: Since Zinc is involved in redox pathways, it should be fine to mention the SOD1 protein (Cu-Zn superoxide dismutase).

Response: We have inserted a statement concerning antioxidant function of Zn (lines 66-67) as quoted below.

“The anti-oxidant enzyme, superoxide dismutase 1 (SOD1), is an example for Zn involvement in cellular defense mechanisms against oxidative stress [19].”

Point 4: Again, concerning the SOD1 protein, it should be fine to mention a recent article concerning the role of Cd in altering the structure and function of SOD1 in human cells (doi: 10.1016/j.redox.2019.101102).

Response: We thank the reviewer for giving us this useful report.  We have discussed findings of this report in the text (lines 167-169) as quoted below. We cited the report as reference # 19.

“It was shown in another study that Cd did not substitute Zn in the antioxidant enzyme, SOD1, but it reduced the activity of SOD1 in human embryonic kidney cells (HEK293T) through MT induction and perturbation of Zn homeostasis [19].”

19. Polykretis, P.; Cencetti, F.; Donati, C.; EnricoLuchinat, E.; Banci, L. Cadmium effects on superoxide dismutase 1 in human cells revealed by NMR. Redox. Biology 2019, 21, 101102.

Point 5: Focusing on UROtsa cells and their clones (UTCd1-d7), Figure 2 shows only differences between UTCd1 and d6. It should be fine to illustrate all the other clones or, at least, to mention their histogram lack in the main text.

Response: As advised, we have now inserted Table 2 that shows data from all seven UROtsa clones (UTCd1-UTCd7).

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript consisted of a review of genetic effects and epigenetic effects of cell zinc and cadmium related transporters and possible relations to carcinogenicity. I suggest minor revision and have only limited comments that should be addressed.

1. Introduction, line 34: Although it is likely that some cadmium is transported in cigarette smoke as the oxide, as stated by the authors, the high transmission of cadmium in cigarette smoke relative to other metals is more consistent with the volatility of the neutral metal than the nonvolatile oxide at the temperature of a cigarette burning coal. See "Pappas RS, Fresquez MR, Watson CH. Cigarette smoke cadmium breakthrough from traditional filters: implications for exposure. J. Anal. Toxicol. 2015;39:45-51." It is likely that just as cadmium's periodic homologue, mercury, is reduced by reducing substances in cigarette smoke, cadmium is possibly also reduced to metallic form rather than being converted to the nonvolatile oxide. The authors should consider revising the statement on the form of cadmium transported in smoke.
2. The authors seemed to focus on the potential role of ZnT1 as a protein that mediates efflux of cellular cadmium, though in line 82, they do mention that it mediates excess zinc efflux as well. Since ZnT1 mediates efflux of both Zn and Cd, it would seem that the cadmium/zinc ratio in the cell would be important. The authors should consider this in rewording some discussion of the role of ZnT1.
3. The authors discuss ZIP8 and ZIP14 as ubiquitous transporters that permit both zinc and cadmium to enter cells on page 2. ZIP8 is downregulated upon exposure of cells to cadmium, preventing further uptake of both zinc and cadmium. One possible carcinogenic mechanism that the authors should consider is that if the cell cadmium/zinc ratio is high, downregulation of ZIP transcripts would not only block further cadmium influx, but zinc as well. This would prevent reversal of the elevated internal cadmium in presence of more favorable levels of zinc. Perhaps this should also be considered in discussion of carcinogenic mechanisms in addition to the effect of cadmium tolerance discussed by the authors. Immediate cell survival at a cost of high internal cadmium/zinc ratio elevating risk of carcinogenicity could be a double edged sword in cell response to cadmium exposures. See Xu, Y.-M.; Gao, Y.-M.; Wu, D.-D.; Yu, F.-Y.; Zang, Z.-S.; Yang, L.; Yao, Y.; Cai, N.-L.; Zhou, Y.; Chiu, J.-F.; et al. Aberrant cytokine secretion and zinc uptake in chronic cadmium-exposed lung epithelial cells. Proteom. Clin. Appl. 2016.
4. Beginning with line 256, there is discussion of effects of cadmium on DNA methylation. Changes in DNA methylation is almost considered a biomarker of chronic cadmium exposure now. The authors should expand on this concept in the review. See Qiao Yi Chen, Thomas DesMarais, Max Costa. 2019. Metals and Mechanisms of
   Carcinogenesis. Annual Review of Pharmacology and Toxicology 59:537–554.

Author Response

Reviewer 2

Comments and Suggestions for Authors

This manuscript consisted of a review of genetic effects and epigenetic effects of cell zinc and cadmium related transporters and possible relations to carcinogenicity. I suggest minor revision and have only limited comments that should be addressed.

Response: We thank the Reviewer for his/her evaluation of our review article and for comments to improve our paper.  We have provided below point-by-point responses to the issues raised.

Point 1: Introduction, line 34: Although it is likely that some cadmium is transported in cigarette smoke as the oxide, as stated by the authors, the high transmission of cadmium in cigarette smoke relative to other metals is more consistent with the volatility of the neutral metal than the nonvolatile oxide at the temperature of a cigarette burning coal. See "Pappas RS, Fresquez MR, Watson CH. Cigarette smoke cadmium breakthrough from traditional filters: implications for exposure. J. Anal. Toxicol. 2015;39:45-51." It is likely that just as cadmium's periodic homologue, mercury, is reduced by reducing substances in cigarette smoke, cadmium is possibly also reduced to metallic form rather than being converted to the nonvolatile oxide. The authors should consider revising the statement on the form of cadmium transported in smoke.

Response: As advised, we have rewritten statements in the Introduction regarding the forms of cadmium in cigarette smoke (lines 34-36) as quoted below. We have cited Pappas et al. 2015 as reference 5.

“Cd exists in cigarette smoke as a non-volatile oxide form (CdO), and a volatile metallic form with high transmission rates [5].”

Point 2: The authors seemed to focus on the potential role of ZnT1 as a protein that mediates efflux of cellular cadmium, though in line 82, they do mention that it mediates excess zinc efflux as well. Since ZnT1 mediates efflux of both Zn and Cd, it would seem that the cadmium/zinc ratio in the cell would be important. The authors should consider this in rewording some discussion of the role of ZnT1.

Response: We inserted statements where appropriate to indicate a Cd-to-Zn ratio as an important contributor to net Cd efflux (lines 299-301) quoted below as an example.

“In theory, however, intracellular Cd-to-Zn ratio would be an important contributor to an efflux of Cd, given that ZnT1 mediates efflux of both Zn and Cd [22, 25, 31].”

Point 3: The authors discuss ZIP8 and ZIP14 as ubiquitous transporters that permit both zinc and cadmium to enter cells on page 2. ZIP8 is downregulated upon exposure of cells to cadmium, preventing further uptake of both zinc and cadmium. One possible carcinogenic mechanism that the authors should consider is that if the cell cadmium/zinc ratio is high, downregulation of ZIP transcripts would not only block further cadmium influx, but zinc as well. This would prevent reversal of the elevated internal cadmium in presence of more favorable levels of zinc. Perhaps this should also be considered in discussion of carcinogenic mechanisms in addition to the effect of cadmium tolerance discussed by the authors. Immediate cell survival at a cost of high internal cadmium/zinc ratio elevating risk of carcinogenicity could be a double edged sword in cell response to cadmium exposures. See Xu, Y.-M.; Gao, Y.-M.; Wu, D.-D.; Yu, F.-Y.; Zang, Z.-S.; Yang, L.; Yao, Y.; Cai, N.-L.; Zhou, Y.; Chiu, J.-F.; et al. Aberrant cytokine secretion and zinc uptake in chronic cadmium-exposed lung epithelial cells. Proteom. Clin. Appl. 2016.

Response:  Please see response to point 4 below.

Point 4: Beginning with line 256, there is discussion of effects of cadmium on DNA methylation. Changes in DNA methylation is almost considered a biomarker of chronic cadmium exposure now. The authors should expand on this concept in the review. See Qiao Yi Chen, Thomas Des Marais, Max Costa. 2019. Metals and Mechanisms of Carcinogenesis. Annual Review of Pharmacology and Toxicology 59:537–554.

Response: We thank the Reviewer for insightful comments and guidance. Using data from suggested references (Xu et al. (2016) and Chen et al. (2019), we have constructed new paragraphs to clarify our central hypothesis regarding Cd tolerance that is acquired through alteration of Zn (Cd) transporters (lines 357-375) as quoted below. We believe that perturbation of Zn homeostasis through changes in expression Zn (Cd) transporter genes is a universal mechanism by which Cd induces malignant cell transformation.  

“Several mechanisms have been postulated to explain how chronic exposure to Cd increases cancer risk. These include oxidative stress, apoptosis resistance, defective DNA damage repair, and altered gene expression [99]. Data from UROtsa cells reviewed herein suggest that chronic exposure to low-level Cd induces cellular adaptive responses involving MT, and Zn (Cd) transporters, notably ZnT1 efflux transporter, to acquire Cd tolerance. This adaptive survival mechanism was seen also in rodent lung epithelial cells in which the ZIP8 gene was downregulated after chronic Cd exposure [100]. However, the diminished ZIP8 expression also caused a reduction in cellular Zn uptake and Zn deficiency [100]. Thus, the immediate cell survival at a cost of high intracellular Cd-to-Zn ratio elevating risk of carcinogenicity could be a double-edged sword in cell response to Cd exposures.

In another study, a continuous exposure to 5 μM Cd²⁺ for 20 wks transformed the HPL-1D, human peripheral lung epithelium, to cancer cells, displaying cancer cell characteristics; decreased expression of the tumor suppressor genes p16 and SLC38A3, increased expression of the oncoproteins KRAS and NRAS and vimentin, the epitheli-al-to-mesenchymal transition marker [101]. Most importantly, transformed HPL-1D cells exhibited Cd tolerance, evident from a diminished Cd accumulation, and increased expression of MT-1A, MT-2A, HO-1, ZnT1, ZnT5 and ZIP8. Therefore, we argue that perturbation of Zn homeostasis through changes in expression Zn²⁺ (Cd²⁺) transporter genes is a universal mechanism by which Cd induces malignant cell transformation.”

99. Chen, Q.Y.; DesMarais, T.; Costa, M. Metals and mechanisms of Carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 2019, 59, 537-554.

100. Xu, Y.-M.; Gao, Y.-M.; Wu, D.-D., Yu, F.-Y.; Zang, Z.-S.;Yang, L.; Yao, Y.; Cai, N.-L.; Zhou, Y.; Chiu, J.-F.; Ching, Y.-P.; 2 , Lau, A.T.Y.  Aberrant cytokine secretion and zinc uptake in chronic cadmium-exposed lung epithelial cells. Proteomics Clin. Appl. 2017, 11, 1600059.

101.Person, R.J.; Tokar, E.J.; Xu, Y.; Orihuela, R.; Ngalame, N.N.O.; Waalkes, M.P. Chronic cadmium exposure in vitro induces cancer cell characteristics in human lung cells. Toxicol. Appl. Pharmacol. 2013, 273, 281-288.

Author Response File: Author Response.pdf