Cadmium chloride (CdCl₂), DEN and carbon tetrachloride (CCl₄) were obtained from MilliporeSigma. Other reagents were of analytical grade.

Male C57BL/6 mice (6 weeks old) were purchased from the Animal Center Institute of Surgery Research of the Third Military Medical University (Chongqing, China). Animals were maintained in specific pathogen-free facilities at Zunyi Medical University (Zunyi, China), under a controlled environment (22±1°C, 50±2% humidity and a 12:12 h light:dark cycle) and free access to purified water and standard laboratory chow. Efforts were made to ameliorate distress and harm to animals by daily monitoring and humane treatment. Animals were adequately cared for, and experimental protocols were in compliance with the Animal Management Guidelines of the Chinese Ministry of Health and approved by the Animal Use and Care Committee of Zunyi Medical University (approval no. 2015–01). The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

After 2 weeks of acclimation, 40 mice (8 weeks of age, weighing 16–19 g) were given the first injection of DEN (90 mg/kg, i.p.). Approximately 80% of mice survived the first DEN injection, and 2 weeks later, the surviving mice were given the second injection of DEN (50 mg/kg, i.p.), according to the protocol (22). At 4 weeks after the initial DEN challenge, mice (12 weeks of age) were orally administered 20% CCl₄, 5 ml/kg, twice a week for 4 months in an attempt to promote liver tumors. At 21 weeks after initial DEN initiation, the mice were randomly divided into the DEN (n=14) and DEN + Cd (n=12) group. There was an additional normal control (n=5) group that did not receive any treatment. Cadmium was administered via the drinking water (1000 ppm) as CdCl₂ from 21–40 weeks according to the previous literature (7,8). Body weights were monitored weekly, and a single ultrasound examination of liver tumor formation was performed at 35–39 weeks after DEN initiation. At the end of the 40-week experiment, the mice were anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and subsequently sacrificed by decapitation. The mouse livers were harvested, and the liver weights and tumor outcomes were recorded. The treatment process is illustrated in Fig. 1A.

The visible tumors were counted and recorded. The tumor incidence (number of tumor-bearing mice), tumor numbers (total tumors found) and tumor size scores (score of 1, tumor size <1 mm; score of 2, tumor size 1–2 mm; and score of 3, tumor size >2 mm) were recorded.

Liver tumor formation was measured using B-mode ultrasound (Vevo^® 2100; Fujifilm VisualSonics, Inc.) with 30-MHz peak frequency linear array transducers (MS400; Fujifilm VisualSonics, Inc.; mean beam frequency range of 22–55 MHz) in a digitized scale as previously described (23). Ultrasound was used to detect tumor incidence and size.

Liver tissues were fixed in 10% buffered formalin for 48 h at room temperature, embedded in paraffin at 60°C and sliced into 3.5-µm-thick sections using a RM2235 microtome (Leica Microsystems GmbH). Sections were deparaffinized in xylene and rehydrated using a gradient of ethanol (100, 95, 85 and 75%). The histological sections were then stained with hematoxylin and eosin at room temperature for 8–10 min and 4–5 sec, respectively. The digitized images of slices were observed via the Olympus image analysis system (light microscope; Olympus Corporation) at ×4 and ×10 magnifications as previously described (24).

Metallothionein (MT) protein retrieval was performed in 10 mmol/l citrate buffer (pH 6.0) at 92°C for 15 min. Endogenous peroxidase was then blocked with 3% H₂O₂ for 15 min at room temperature. The tissues were then incubated with 10% goat non-immune serum (Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Subsequently, sections were incubated with MT primary antibodies (cat. no. ab12228, 1:100; Abcam) overnight at 4°C. After washing with PBS for 5 min, slides were incubated with HRP-conjugated anti-mouse secondary antibody (1:1,000; cat. no. A0216; Beyotime Institute of Biotechnology) for 1 h at room temperature following the protocol associated with the SABC Detection System (Beyotime). Finally, sections were stained with DAB and counterstained with hematoxylin for 5 min at room temperature. The digitized images of slices were observed via the LEICA image analysis system (Leica Microsystems GmbH) at ×10 magnification. The expression of proteins was quantitatively measured by Image ProPlus 6.0 to obtain the positive staining-integral optical density/area (density mean). A total of four discontinuous areas were used to analyze the expression levels of protein in a blinded fashion as previously described (24).

Total RNA was extracted from tissues with TRIzol^® (Takara Biotechnology Co., Ltd.) and reverse transcribed according to the manufacturer's protocol into cDNA using the PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.). qPCR was performed utilizing the iQ™ SYBR Green Supermix (Bio-Rad Laboratories, Inc.). The PCR cycling conditions were 94°C for 3 min, followed by 40 cycles of 94°C for 15 sec, 60°C for 20 sec and 72°C for 40 sec. Data were normalized to β-actin and expressed using the comparative Cq method (23,25,26). Primers for the mouse genes are shown in Table I.

Data are presented as the mean ± SE and were analyzed by one-way ANOVA, followed by Ducan's multiple comparison test using SigmaPlot (Systat Sofeware, Inc.) version 14. For tumor incidence, Fisher's exact test was performed for the contingency table, while for tumor number and tumor score, data were analyzed with the Kruskal-Wallis test, followed by Dunn's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

After the initial DEN (90 mg/kg, i.p.) administration, 80% of mice survived within 7 days, with a ~20% reduction in body weight. After the second DEN injection, 95% of mice survived, with a ~15% reduction in body weight. Animal body weights slowly recovered afterwards over time. CCl₄ promotion (20%, 5 ml/kg, p.o., twice/week) was started at week 4 post-DEN injection and continued for 4 months. Cd was administered from 21–40 weeks after DEN initiation through the drinking water (1000 ppm) for 19 weeks. All mice survived Cd treatment. Ultrasound was randomly performed on a few mice at week 36 post-DEN injection (15 weeks after Cd treatment), 4 times, until week 40 post-DEN initiation (19 weeks after Cd treatment). The animal body weights after Cd intervention are shown in Fig. 1B.

Small animal ultrasound analysis was used to monitor tumor formation 30 weeks after DEN injection, but no tumors were detected. At 36 weeks after DEN injection, liver tumors with small sizes were detected at an ~40% incidence rate. At week 40 after DEN initiation, a 65% tumor incidence rate was recorded. Fig. 2 shows representative ultrasound images of DEN-induced liver tumors at 40 weeks after DEN initiation in control, DEN- and DEN + Cd-treated mice. In the control mice, the ultrasound showed normal liver, while in the DEN-treated mice, the liver density was increased (arrows), indicative of liver tumors. In the DEN + Cd-treated mice, only one mouse showed a detectable tumor with decreased density compared with non-tumor tissues.

At the end of experiment, the mice were euthanized and their livers were collected and weighed, and the liver/body ratios were calculated. Fig. 3 shows the liver/body weight ratios of the three groups. DEN increased the liver/body weight ratio from 48.5 to 53.4 mg/g, while DEN + Cd further increased the liver/body weight ratio to 55.9 mg/g, which were significantly higher results compared with the controls.

Representative gross liver images are shown in Fig. 4, where liver tumors were detected in DEN-treated mice, no tumors were found in control mice and only a few tumors were found in DEN + Cd-treated mice.

The tumor outcomes, recorded from blinded observations, are listed in Table II. In total, 10 out of 14 DEN-treated mice had tumors, with a tumor incidence of 71%, and only 2 out of 12 DEN + Cd-treated mice had tumors, with a tumor incidence of 17%; a total of 15 tumors were found in DEN-treated mice, but only 2 tumors in DEN + Cd-treated mice. Some tumors in the DEN-treated mice were large, and the tumor score was 22, while in DEN + Cd-treated mice, the score was 3. The tumor number and tumor score of the DEN + Cd group were statistically significant compared with the DEN group.

Fig. 5 shows representative histology and immunochemistry images of the tissues in the different groups. Liver tumors were observed in DEN-treated mice, and hepatocellular degeneration was evident in both DEN- and DEN + Cd-treated mice. In DEN-treated livers, MT staining was not present in the liver tumors, but was strongly present in the tissues surrounding the tumors. DEN + Cd-treated mice exhibited increased intensity of the MT stain.

Fig. 6 shows the expression of liver genes in the control mice (n=5), and in the mice treated with DEN (n=14) and DEN + Cd (n=12), with controls set at 100%. DEN treatment increased the expression of α-fetoprotein (AFP) over 2-fold (242% of control); however, due to huge individual variance, the difference was not statistically significant. DEN + Cd treatment increased the expression to 112% of the control. The expression of MT-2 was slightly decreased by DEN treatment (73% of the control), but significantly increased by DEN + Cd treatment by almost 3-fold (294% of the control). The expression of tumor necrosis factor α (TNFα) was slightly increased by DEN treatment (142% of the control), but significantly increased by DEN + Cd treatment (224% of the control).