Figure 1. Dose-response relationship between dietary Zn deficiency and esophageal cancer development.

A. Study design - 4-week-old rats were fed diets containing 3, 6, 12, or 60 mg Zn/kg, forming marked-ZD (ZD3), moderate-ZD (ZD6), mild-ZD (ZD-12), and Zn-sufficient (ZS) groups (n = 47-49/group). After 5 weeks, NMBA-treated tumor (T) groups received 4 intragastric NMBA doses, once a week for 4 consecutive weeks (2 mg/kg body weight; n = 26-27 rats/group); control groups were NMBA-untreated (n = 10 rats/group). The study was concluded 17 weeks after the 1st NMBA dose. B. At 5 weeks after dietary regimen, immunohistochemical analysis showing abundant and strong PCNA-positive nuclei (red, 3-amino-9-ethylcarbazole substrate-chromogen) in in multiple cell layers (arrow head) in proliferative ZD3, ZD6, and ZD12 esophageal epithelia vs few PCNA-positive nuclei in nonproliferative ZS control (scale bars = 100 μm, x100 magnification). Open rectangles are insets illustrated in panels directly below (scale bars = 25 μm, x400 magnification). PCNA-LI (labeling index, %) was significantly higher in ZD3 vs ZS, ZD6 vs ZS, ZD12 vs ZS group, and significantly higher in ZD3 vs ZD6, and ZD3 vs ZD12 group (***P < 0.001, ⁺⁺⁺P < 0.001, n = 12 rats/group). At 22-weeks, ZD3, ZD6, and ZD12 esophagi showed focal hyperplastic lesions (arrow head) with abundant PCNA-positive nuclei vs nonproliferative ZS control (scale bars = 100 μm, x100 magnification). Open rectangles are insets illustrated in panels directly below (scale bars = 25 μm, x400 magnification). C. Tumor endpoint (n = 26-27 rats/group): body weight (g), serum Zn levels (μg/100 ml), esophageal tumor multiplicity (number of tumors/esophagus), tumor incidence (%), and esophageal squamous cell carcinoma (ESCC) incidence (%), (ZD tumor group vs ZST control: ***P < 0.001, **P < 0.01, *P < 0.05. ZD6T vs ZD3T and ZD12T vs ZD3T: ⁺⁺⁺P < 0.001, ⁺⁺P < 0.01, ⁺P < 0.05). Error bars represent standard deviation.