Toxicity of Organic and Inorganic Nickel in Pancreatic Cell Cultures: Comparison to Cadmium

Nickel compounds are Group 1 carcinogens and possibly cancer-causing in the pancreas. We examined the toxicity of nickel in both 2-D and 3-D pancreatic cell cultures, to determine the LD50 for organic and inorganic nickel in normal and cancerous cells. Assays with cadmium chloride were performed to be a comparison to potential nickel-induced toxicity. Cells were exposed to twelve concentrations of NiCl2 or Ni-(Ac)2 for 48h (2-D), or six concentrations for 48 hours (3-D). There was a significant (P=0.0016) difference between HPNE and AsPC-1 LD50 values after cadmium exposure, at 69.9 μM and 29.2 μM, respectively. Neither form of nickel exhibited toxicity in 2-D or 3-D cultures, but after 48h, changes in spheroid morphology were observed. The inability of Ni to reduce viable cell numbers suggests a toxic mechanism that differs from cadmium, also a Group 1 carcinogen. The cell microenvironment was not a factor in nickel toxicity with no changes in viable cells in either 2-D or 3-D cultures. These studies only examined cytotoxicity, and not genotoxicity, a potential mechanism of nickel carcinogenicity. Alterations in DNA function or the expression of apoptotic proteins/processes would take longer to manifest. Current work focuses on cellular changes following extended nickel exposure.


Introduction
Nickel (Ni) is a ubiquitous metal found naturally in the environment and associated with manufacturing and commercial processes (1). Our understanding of Ni-related toxicity has linked Ni exposure to multiple disease states (2,3). Exposure to Ni may also occur by consumption of contaminated foods or agricultural products (4,5). Besides contaminating sources such as commercial items, a major source of Ni is through vaping and the inhalation of Ni contaminated vapor (6,7). Numerous reports have linked exposure to Ni through processes like welding, electroplating, and painting (8)(9)(10)(11). Of the metals that fall within the broad category of 'heavy metals,' Ni has been understudied compared to metals like mercury, lead, cadmium (Cd), and manganese. Cd and Ni are often found together in the environment. Both metals have been shown to bind to soil humic substances, which facilitates the joint movement of the metals through the environment (12). Ni has presented an insidious health risk with symptoms not presenting for months/years after exposure. It has been shown that Ni accumulates in different organ systems at different rates. The lungs and thyroid are areas rich (140-170 μg/kg) in Ni, whereas the pancreas has one of the lowest Ni content at 34 μg/kg (range of 7-71 μg/kg) in humans (13). Ni is not readily cleared from the body and may bioaccumulate, posing additional health risks. Recent evidence suggests that Ni exposure may result in endocrine-related changes, including tumor formation (14)(15)(16). Similar to Cd, Ni has been suspected of acting as a metalloestrogen, activating the estrogen ERα receptor, but overall, the information is scarce (17). A critical action of Ni-mediated toxicity has been reported to be the generation of free radicals and the promotion of oxidative stress (18)(19)(20), leading to an increase in lipid peroxidation (21).
Pancreatic cancer (PC) is one of the most lethal human cancers and an important cause of cancer-associated-mortalities worldwide (22,23). The primary environmental factors associated with PC so far are inhalation of cigarette smoke, exposure to mutagenic nitrosamines, chlorinated hydrocarbon solvents, and heavy metals (24). Involuntary exposure to heavy metals in humans, particular attention to Cd, is important due to its abundance as an occupational and environmental pollutant (25). Previous studies have linked Cd exposure and PC development due to Cd accumulation in the pancreas (26,27). We chose to use Cd as a form of internal control based on its known ability to cause cell death and promote pancreatic cancer pathogenesis. Global health organizations have listed both Cd and Ni as class I carcinogens (28,29). Reviews from different groups have outlined the mechanisms associated with metal-and metalloid-induced toxicity leading to cancer development (16,29,30). A positive correlation between Cd concentration and the incidence of PC has been reported for samples taken from toenails (31). However, there was a negative correlation between Ni concentration and PC incidence (31). A similar negative correlation for Ni concentration in the pancreas has been reported in early pancreatitis (32). In cases of PC, Ni is present in pancreatic juices but does not directly correlate with cancer incidence; instead elevated Ni content correlates with elevated chromium in the pancreatic juice (28,33). This secondary correlation suggests an indirect involvement of Ni.
Historically available technology limited cell culture work. Work in two dimensions (2-D) predominates the literature. Recently, technology has permitted the development of cell culture work in three dimensions (3-D). Multiple platforms exist for developing 3-D cultures, including the use of specialized 96-well plates with concave wells, which is our model system (34)(35)(36). The technologies available are outlined in numerous reviews, and these authors stress the importance of the 3-D model in capturing the complexity associated with the naturally occurring tumor environment (37)(38)(39)(40). Since 3-D methodologies are relatively new, most of the literature published is over the last five years. Literature describing the use of 3-D culture for the study of PC is scarce. There have been no reports describing HPNE cells in 3-D culture, and publications describing the AsPC-1 cells in culture are few (34,41,42). This lack of information is one of the foundations for the importance of the studies presented here.
To best determine toxicity following metal exposure, the appropriate cell microenvironment was utilized. There are limitations with the conventional 2-D culture methods, with 3-D cultures being touted as a good alternative to 2-D culture models. We aim is to develop and use assay conditions that further our understanding of PC biology in 3-D cultures and establish criteria for studying toxic mixtures of environmental toxicants. The goal of these studies is to compare the effects of nickel chloride (NiCl2) or nickel acetate (Ni-(Ac)2) on the viability of HPNE and AsPC-1 pancreatic cells. Two growth methodologies are utilized (2-D v. 3-D) to examine the effects of cell microenvironment on the cellular response to NiCl2 exposure.
Collectively, these studies are the first to compare the development and functionality of spheroids for the HPNE and AsPC-1 cell lines and examine Ni-mediated toxicity in both 2-D and 3-D cultures. As the current methodology improves, the use of spheroids to accurately mimic the milieu of the tumor will be vital.

Methods
Cell Lines and Cell Culture Maintenance: Cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). Pancreas hTERT-HPNE ("Human Pancreatic Nestin-Expressing"; ATCC® CRL-4023™, immortalized control pancreatic cells -referred to as 'HPNE') and AsPC-1 (ATCC® CRL-1682™, pancreatic tumor cells) were grown and maintained, as described in ATCC protocols. Cells were maintained at 37°C with 5% CO2. hTERT-HPNE cell line is a control human pancreatic ductal cells that were transfected with the hTERT gene using the retroviral expression vector, pBABEpuro. Transfected cells did not senesce and continued to proliferate (43).
Cell Treatment: Cells were initially grown in a 25cm 2 flask to near confluence having reached their exponential growth phase. Cells were then trypsinized, removed, and subjected to Trypan Blue cell counting using a Corning® CytoSMART cell counter. Cells were diluted in growth media to a final concentration of 10 5 cells/mL before the addition of 100 μL of cell suspension per well. Cells were returned to the incubator for 24h, after which the media was removed, and assay media (MEM with 1% FBS; no phenol) was added with the appropriate concentration of NiCl2 or Ni-(Ac)2 and the exposure to Ni was continued for 48h.
3-D Spheroid Culture Growth: Cells were maintained using conventional methods and grown in a 25cm 2 flask (approximately 80% confluent; the exponential part of the growth phase). Cells were trypsinized for removal from the flask, and media was added to yield a final concentration of approximately 2.5x10 4 cells/well. To promote orderly spheroid growth, clumps of cells must be removed from suspension. To facilitate this, suspensions were passed through a 40-μm cell strainer to yield a homogenous population of single cells. A 100 μL aliquot of the 'single-cell' suspension was placed in each well and spheroid formation was observed at 24h and 48h. Cells were grown in their normal growth media using specialized concave microsphere plates (Corning, Model #4515).
Cell Viability Measurements in 2-D cultures -MTT assay: MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) stock solutions were prepared by adding sterile PBS to a final concentration of 12.5 mM MTT. Stock MTT was diluted in-well by the addition of 10 µL of MTT stock to each well (1.1 mM final concentration). Plates were returned to the incubator for 4 hours. After incubation, 50 µL of DMSO was added to each well to solubilize MTT crystals, and the plates were returned to the incubator for 10 min. Absorbance was measured using a BioTek plate reader at 540 nm.
Cell Viability Measurements in 3-D Spheroids: Only the AsPC-1 cells were utilized in this study due to the improved spheroid formation in the tumor cells compared to HPNE cells. Viability measurements were obtained using the CellTiter-Glo®-3D assay kit (Promega) as described in the manufacturer's guidelines and protocol. Following exposure to NiCl2 for 48h, assays were performed following the manufacturer protocol. Incubation was carried out at room temperature (22°C) for 25 minutes, and the resulting ATP-related luminescence was measured at 590 nm using a BioTek plate reader.
Statistical Analysis: All data were analyzed using the non-normalized data using a one-way ANOVA, followed by Dunnett's-test for posthoc comparisons of treatment groups to control. LC50 values were analyzed by a two-tail t-test. All analyses and graphics were generated using GraphPad Prism (v8.4.3; GraphPad, San Diego, CA). Data are expressed as mean ± SEM of (n=3-8). Alpha-level significance was set at α < 0.05.

3-D Spheroid
Culture Growth: Both HPNE control cells and AsPC-1 tumor cells were grown in the specialized concave culture plates designed specifically for spheroid development (Figure 1). It was clear from the images and dilutions at both 24h and 48h incubation times that the AsPC-1 tumor cells formed spheroids with greater definition than HPNE cells. At the maximum (10 4 cells/well; 100%) concentration, a clearly defined spheroid was evident after 24h in the AsPC-1 group. HPNE cells didn't form a conventional spheroid but instead formed a dense, asymmetrical 'clump.' Further examining cell dilutions, 50% (5,000 cells/well), 25% (2,500 cells/well), 12.5% (1,250 cells/well) and 6.25% (625 cells/well) demonstrated that the AsPC-1 group formed spheroids even at the lowest concentration of cells, but the spheroid was less defined than with dilutions higher than 25%. Determination of LC50 values for Cd and Ni: Incubating HPNE and AsPC-1 cells with increasing concentrations of CdCl2 (100 nM -10 mM) resulted in reduced viability at the higher concentrations ( Figure 2). Using the Least Squares Fit model, nonlinear regression analysis resulted in a converging fit for HPNE (R 2 = 0.9814) and AsPC-1 (R 2 = 0.9496) cells and provided LC50 for each run. To determine if 'within group' variance contributes to the 'between-group' differences, the D'Agostino-Pearson (K2) test was utilized, and both HPNE (K2 = 0.2595) and AsPC-1 cells (K2 = 0.4752) passed the normality test. Comparing the LC50 values (Figure 2, inset) revealed HPNE cells exhibited a significantly (p = 0.0016) higher LC50 value (69.9 ± 9.1 μM) compared to AsPC-1 cells (29.2 ± 2.7 μM). Ni-mediated changes in viability in AsPC-1 spheroid cultures: AsPC-1 cells were grown in spheroid culture as described in the methods. Plates were returned to the incubator for 24h at which time the media was removed, assay media added, and the photomicrograph (10x) taken, representing the 0h time point (Figure 4, left panel). Note the yellow circle on each panel encompassing the spheroid. The same circle is positioned on the right side of the image, 48h after exposure to 50 μM NiCl2 or Ni-(Ac)2. We observed that after 48h of exposure, there did not appear to be a reduction in cell number, but the spheroids lost their organized structure, starting on the outer layers of the spheroid. Measuring the cell viability in the spheroid, we observed no noticeable reduction in viability or ATP release following one-way ANOVA analysis examining the effect of treatment ( Figure 4, right panel). The rationale for using 50 μM NiCl2 or Ni-(Ac)2 was to examine the low concentration effects. By modifying the equation for fractional occupancy (eq. 1) for LC50, we can derive an estimate of the lethality based on the metal concentration and its LC50 value.
Using this modified equation, we can estimate the cell lethality for 50 μM NiCl2 to be 5.5%, and for Ni-(Ac)2, the lethality is 8.6%. In both instances, low lethality would be within the error of the viability assay. Although viability was low, exposure to Ni appeared to disrupt the formation of the spheroid.

Discussion
Our previous studies have established a body of evidence that strongly suggests that Cd is toxic in the pancreas and may lead to the development of pancreatic cancer, and other physiological dysfunctions such as insulin dysregulation and thyroid dysfunction (44)(45)(46)(47). Ni and Ni-containing compounds have not been studied to the same extent as Cd. As a result, the primary aim of this study was to begin an investigation into Nimediated toxicity in the pancreas. Also, we initiated studies to compare the actions of toxicants in 2-D versus 3-D cell culture. There are subtle differences between toxicant responses in the two systems (35,36). In particular, the pancreas has become an organ system critical for the development of 3-D model systems to be used in cancer studies (42). To date, few studies have utilized 3-D cultures in the study of PC, and none have examined Ni's effects following short-term exposure.
Using Cd toxicity as an internal assay control to assess Ni-mediated toxicity was done to validate our assay conditions. The determination of LC50 values for Cd was similar to what we have previously reported (47). Contrary to our findings with Cd, the toxicity of Ni was minimal in the initial 2-D studies. Our LC50 values were 10-20-fold higher than for Cd (> 500 μM). The present studies lay the foundation for a future examination into the cellular effects of Ni-mediated toxicity.
On the surface, the relatively low lethality associated with either NiCl2 or Ni-(Ac)2 exposure for 48h would suggest a generalized lack of toxicity associated with Ni. These findings are supported by current literature describing Ni-mediated effects in a variety of model systems. In rodent β-cells, exposure to NiCl2 for 24h did not elicit a significant reduction in viability until the test concentration exceeded 1 mM (15). In mouse and human cell lines, NiCl2 exposure elicits an interesting profile of toxicity. In mouse cells, lower concentrations of Ni (100-200 μM) increased viability compared to control values, but at 600-800 μM, there was a nearly 50% reduction in viability (48). In human HepG2 cells, increasing Ni concentration, resulted in decreasing viability, with a 50% reduction at 400-600 μM (48). In human lymphocytes, significant reductions in viability were not observed until a concentration of 3 mM was exceeded (18). The viability reports in the literature support our findings of LC50 values of >500 μM dependent on the cell line and the chemical form of Ni (organic or inorganic). Although not overtly toxic, further studies will be needed to investigate long-term exposure to Ni.
One of the mechanisms of Ni-induced toxicity is the promotion of oxidative stress. When the current information is examined, it is evident that Ni's ability to induce free radical generation is about as potent as its lethality. In vivo models using injections of moderately high Ni doses resulted in increased expression/activity of various biomarkers. These biomarkers are associated with oxidative stress such as alkaline phosphatase, aspartate transaminase, oxidized lipids, malondialdehyde, or glutathione reduction (20,49,50). A study by Chen et al. (18) generation of free radicals following NiCl2 administration showed that dichlorofluorescein fluorescence was not elevated until a concentration of 1 mM was used. The use of high concentrations suggests Ni is a weak promoter of oxidative stress. Exposure duration was only 1h, which for an in vitro assay would be sufficient for significant radical generation, only yielded a 3-fold increase in oxidative stress over baseline (18). Even if Ni is a weak free radical generator, the in vivo studies leave open the possibility that a second pathway is involved. Ni administration may work through multiple pathways to increase oxidative stress.
Although Ni is considered a carcinogen, it has been shown to exhibit an inverse correlation between Ni content and the incidence of PC (31,32). One report demonstrated a positive correlation between Ni content and PC involving KRAS mutations (51). There is a mounting body of evidence that supports Ni involvement in an array of pancreasrelated dysfunction. Application of Cd to β-cells elicits responses mediated by the L-type (long-lasting) calcium channels, whereas Ni works through the transient T-type calcium channels (52). When Ni is applied to β-cells, there was no response in calcium flux or insulin release suggesting a lack of T-type channels on β-cells. Yet, there is a mechanism for Ni-induced damage in the pancreas. Parental exposure to NiCl2 results in an increase in oxidative stress markers in the pancreas (14). Ni itself appears to be a 'hyperglycemic' metal, a metal that promotes blood glucose elevation, either by reducing insulin release, blocking insulin action, or promoting glycogen breakdown/de novo glucose synthesis. The in vivo result of Ni exposure is hyperglycemia resembling diabetes and may involve other mediators (21,53,54).
Collectively, these studies provide the foundation and the initial set of data that we can build from moving forward in our study of Ni toxicity and its involvement in PC development. The use of 3-D cultures is critical for furthering our understanding of metalor Ni-induced toxicity and PC development. The tumor cells tend to form very distinct and orderly spheroids. In the concave well model, control HPNE cells tended to form more of a dense cluster that doesn't resemble the archetypical spheroid. A different model system may be needed, with the use of Matrigel® or magnetic microbeads as potential options that would by-pass the need for the cells to initiate their spheroid formation.
Initially, it appears that our LC50 values were low, but when compared to the literature, Ni is not a highly lethal metal. Concentrations > 500 μM are needed to elicit an increase in free radical formation or cell death. Interestingly, it appears that Ni may be involved with other metals or toxicants, and the relationship between the different toxicants will direct the toxic outcome. The theory of chemical mixture toxicity needs to be explored further. Future studies are already underway characterizing cellular changes involving apoptotic pathways following Ni exposure. Another interesting avenue to pursue is Ni exposure resulting in epigenetic changes (55)(56)(57)(58). As technology continues to develop and advance, we will continue to advance our understanding of Ni-mediated toxicity and Ni involvement in PC development.