Introduction

Copper oxychloride [3Cu(OH)2·CuCl2] is a common broad spectrum fungicide used to protect vegetables and fruits against diseases such as Venturia inaequalis, Taphrina deformans, Coryneum beijerinckii, Xanthomonas campestris pv, Vesicatoria, Phytophthora infestans, Alternaria solani, and Deuterophoma tracheiphila (Pérez-Rodríguez et al. 2013; Al-Assiuty et al. 2014).

Copper is a heavy metal that can be accumulated in various tissues of all living species (Snyman et al. 2004; Matache et al. 2013; Hurwitz et al. 2014). Its continuous application may lead to copper accumulation in crops, soil, and water (Chen et al. 1997; Masaka and Muunganirwa 2007) that consequently leads to human and animals to be exposed to copper through the food chain (La Pera et al. 2008), causing diseases such as kidney dysfunction, hepatocellular neoplasia, and hemolytic anemia (Waheed et al. 2013; Hurwitz et al. 2014; Ciji and Bijoy Nandan 2014).

Chronic copper poisoning can also cause weakness, abdominal pain, headache, dizziness, leg and back pain, anemia, and chronic kidney and liver damage in humans (Gunay et al. 2006; Hloch and Charvát 2012).

There are a few studies that have confirmed the genotoxic and cytotoxic effects of copper oxychloride in test animals using chromosome aberration and molecular toxicity tests (Pirtskhelani et al. 2008) or random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) (Atienzar et al. 2001; Gupta and Sarin 2009). To our best knowledge, so far none has examined its genotoxic effects on human lymphocytes as the most relevant markers of human exposure (Madle et al. 1993; Buyukleyla and Rencuzogullari 2009; Sevindik and Rencuzogullari 2014). The aim of this study was to fill this gap by investigating the genotoxic effects of copper oxychloride using widely adopted methods to detect DNA damage in humans, namely chromosome aberration (CA), micronucleus (MN), and RAPD-PCR tests (Albertini et al. 2000; Norppa and Falck 2003; Atienzar and Jha 2006; Kocaman and Topaktaş 2010).

Materials and methods

The test substance copper oxychloride was obtained in the commercial formulation that contains min 95 % copper oxychloride with min 51.4 % of the suspended active ingredient Cu (514 g Cu/L) (Tarem Inc., Delaware, DE, USA). The formula and other properties of the product are given in Table 1.

Table 1 The formula and properties of copper oxychloride
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Chromosome aberration and micronucleus test

Chromosome aberration test was applied following the method described by Evans (1984) and the MN test was used as described by Fenech (2000) and Kirsch-Volders et al. (2003) with minor modifications. We also followed the IPCS guidelines (Albertini et al. 2000) and obtained the approval from the Ethics Committee of Adiyaman University (registered under no. 19.03.2013/03-01.1) in accordance with the Declaration of Helsinki.

To prepare the lymphocytes for CA testing, we added 0.2 mL of heparinized whole blood from five healthy, young donors (three females and two males, all non-smokers, aged 21–22) to 2.5 mL chromosome medium B (Biochrom, Berlin, Germany, F5023) and the culture was incubated at 37 °C for 72 h. The non-cytotoxic copper oxychloride concentrations were established in preliminary study. Cultured cells were treated with 3, 6, and 12 μg/mL concentrations of copper oxychloride which was dissolved in sterile distilled water as described by the manufacturer, for 24 and 48 h. Untreated blood samples and samples treated with 0.22 μg/mL ethyl methanesulfonate (EMS, Sigma, M0880) were used as negative and positive control, respectively. After colchicine treatment, 2 h before harvesting, the cells were treated with a hypotonic solution of 0.4 % KCl for 5 min then fixed three times in a mixture of methanol and glacial acetic acid (3:1). Air dried slides were stained with 5 % Giemsa stain prepared with Sorensen’s buffer for CA test following the standard methods (Evans 1984).

Chromosome aberration frequency and the percentage of abnormal cells (AC) were determined in 100 well-spread metaphases per donor (totaling 500 metaphases per concentration). Gaps were not counted as CA (Mace et al. 1978). Aberrations were classified according to the International System for Human Cytogenetic Nomenclature (ISCN) (Paz-y-Miño et al. 2002). The mitotic index (MI) was also determined by scoring 3000 cells from each donor. The MI detects delaying effects of chemicals on the G2 phase of the cell cycle (Evans 1984).

To score the number of micronuclei in binucleated cells, we used the cytochalasin B-induced cytokinesis block (Fenech 2000; Kirsch-Volders et al. 2003) by adding 0.2 mL of whole blood from each donor to the culture medium and treating the cultures with 3, 6, and 12 μg/mL of copper oxychloride solution for 24 and 48 h. Again, we used negative and positive control as described above. Six μg/mL of cytochalasin B (Sigma, St. Louis, MO, USA, C6762) was added to the cultures at hour of incubation in order to block cytokinesis. After 24-hour incubation with cytochalasin B at 37 °C, the lymphocytes were initially treated with 0.4 % KCl for 2 min then harvested by centrifugation (at 1200 rpm for 10 min). The harvested lymphocytes were fixed once with a mixture of methanol:glacial acetic acid (5:1) and an equal amount of 0.9 % NaCl, and then fixed twice with only methanol:glacial acetic acid (5:1). Air dried slides were then stained with 5 % Giemsa prepared in Sorensen’s buffer (Rencuzogullari et al. 2004). For each subject, 2000 binucleated cells were scored for MN frequency and a total of 1000 viable cells for the frequency of cells with 1, 2, 3, or 4 nuclei to calculate the nuclear division index (NDI) using the formula: NDI = (M1) + (2 × M2) + (3 × M3) + (4 × M4)/N (Fenech 2000), where M1–M4 represents the number of cells with one to four micronuclei and N the total number of viable cells scored.

Statistical analysis

To determine the statistical significance for CA, AC, and MN counts we used the one-way analysis of variance (ANOVA). Correlation and regression coefficients were used to determine dose–response relationships. For all statistical analyses we used MINITAB 14 version (Minitab Ltd., UK). The P value <0.05 was considered significant.

PCR/RAPD test

To determine molecular genotoxicity of copper oxychloride in human lymphocytes with the RAPD method we used 10 oligonucleotide primers (Table 2) with variable GC base pair proportions, as described earlier by Atienzar and Jha (2006) with minor modifications.

Table 2 Primer sequences used for RAPD
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We added peripheral blood from two healthy donors (one male and one female, both non-smokers, aged 21) to chromosome medium and incubated the culture at 37 °C for 72 h. The cultured cells were then treated with 3, 6 and 12 μg/mL concentrations of copper oxychloride solution 24 and 48 h. In addition to the negative and positive control cultures described above (untreated and EMS-treated, respectively), we used a second positive control culture treated with 2 μg/mL sodium azide (SA, Merck, Darmstadt, Germany, 106688). Ethyl methanesulfonate was used to cause frameshift mutation and SA base substitution (single-point mutation). At the end of the exposure period, the cultured cells were washed twice in sterile isotonic solution (0.9 % NaCl), and genomic DNA was isolated using the High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s protocol (http://www.roche-applied-science.com).

Thirty-five RAPD-PCR reactions were performed with a Veriti 96-well thermal cycler (Applied Biosystems, Foster City, CA, USA) using the 10 oligonucleotide primers. PCR reaction mixtures (25 μL of total volume) consisted of the PCR buffer 1× [10 mM Tris–HCl, pH 8.8 and 50 mM KCl], 3 mM MgCl2, 2.5 U/μL of Taq DNA polymerase (Fermantas, Vilnius, Lithuania, SB38), 1.25 μM of each dNTPs (Fermantas, RO191), 4 pmol of each primer, and 50 ng of the DNA sample. PCR conditions were used as follows: an initial denaturation at 95 °C for 5 min, was followed by 35 cycles at 95 °C for 1 min, at 34 °C for 1 min and at 72 °C for 2 min, and a final extension at 72 °C for 10 min. PCR products were electrophoresed on 2 % agarose gel containing 0.5 μg/mL ethidium bromide and visualized under UV illumination. Fragment sizes were confirmed with DNA reference bands (DNA ladder, 100 bp) (Thermo Scientific, Waltham, MA, SM0321).

RAPD profiles and statistical analysis

All amplified bands in untreated control were scored as 0 and new and missing bands in treated groups were scored as 1. Bands in the treated groups that were the same as in control were scored as 0. By scoring the new and missing bands we were able to detect polymorphisms in RAPD profiles. The scores were then statistically analyzed using Student’s t test. The genomic template stability (GTS) rate was calculated using the following formula: GTS = (1 − y/z) × 100, where y is the total number of polymorphic bands in the treated groups (total number of new and missing bands) and z is the total number of bands in negative control, as described by Aras et al. (2011). Changes in RAPD profiles decreased the GTS rate, which indicated to genotoxic effects.

Results

Treatment with copper oxychloride significantly increased the percentage of CA and AC in human lymphocytes at all concentrations of all treatment periods when compared to negative control (Table 3). The effect was dose-dependent for CA in both 24 and 48-hour exposure (r = 0.998, P = 0.042 and r = 0.997, P = 0.048, respectively) and for AC in 24-hour exposure (r = 0.999, P = 0.020) (Fig. 1).

Table 3 Structural chromosome aberrations and percentage of abnormal cells in cultured human lymphocytes treated with copper oxychloride
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Fig. 1
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The percentage of chromosome aberrations and abnormal cells in cultured human lymphocytes treated with CuOCl for 24 and 48 h

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Copper oxychloride also significantly increased the MN frequency and the percentage of micronucleated binuclear cells (MNBN) at all concentrations of all treatment periods when compared to negative control (Table 4). However, the effect was not dose-dependent (Fig. 2).

Table 4 The frequency of micronucleus (MN) and micronucleated binuclear (MNBN) cells in cultured human lymphocytes treated with copper oxychloride
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Fig. 2
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The percentage of micronucleus (MN) and micronucleated binuclear (MNBN) cells in cultured human lymphocytes treated with CuOCl for 24 and 48 h

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Copper oxychloride decreased the MI at only the highest concentration (12 µg/mL) and decreased the NDI at the two highest concentrations (6 and 12 µg/mL) in both exposure periods (Table 5). Again, the effect was not dose-dependent for either index (Fig. 3).

Table 5 The mitotic index (MI) and nuclear division index (NDI) in cultured human lymphocytes treated with copper oxychloride
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Fig. 3
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The MI and NDI in cultured human lymphocytes treated with CuOCl for 24 and 48 h

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The significant increase in total polymorphic bands, observed for all concentrations and treatment periods, was also not dose-dependent (Table 6). The GTS rate also significantly increased in all concentrations and treatment periods but not in a dose-dependent manner (Table 7; Fig. 4).

Table 6 The RAPD bands profile in female and male DNA in human cells exposed to copper oxychloride
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Table 7 Genomic template stability rate of the male and female DNA in human cells exposed to copper oxychloride
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Fig. 4
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The GTS rate of the male and female DNA in cultured human lymphocytes treated with CuOCl for 24 and 48 h

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Discussion

As our study was the first to investigate the genotoxic and cytotoxic effects of copper oxychloride in human lymphocytes, we were not able to compare our findings with similar studies. However, Pirtskhelani et al. (2008) reported a strong genotoxic effect of copper oxychloride in mice and Shivanandappa et al. (1983) reported testicular atrophy in Gallus domesticus fed with acute doses of copper oxychloride. In addition, copper oxychloride caused infertility in some simple animals by decreasing the number of oocytes (Helling et al. 2000; Snyman et al. 2004, 2009). As for studies in humans, several have confirmed the genotoxic and mutagenic effects of copper oxide nanoparticles, which are used in a variety of industries (Ahamed et al. 2010; Wang et al. 2012; Alarifi et al. 2013; Di Bucchianico et al., 2013; Akhtar et al. 2013).

The use of pesticides containing heavy metals such as copper may lead to their transfer to humans through the food chain (Chen et al. 1997; Masaka and Muunganirwa 2007; La Pera et al. 2008; Pose et al. 2009). Waheed et al. (2013) reported that high accumulation of Cu in various organs of herbivorous and carnivorous edible fish may have increased the risk of metal toxicity in humans. Our tested formulation contained other heavy metals such as cadmium, iron, and lead, but in such trace amounts (0.1, 0.01 and 10 ppm, respectively) that they could not affect our findings.

In our study, all copper oxychloride concentrations increased the frequency of chromatid and chromosome breaks and the number polymorphisms and decreased cell proliferation. The resulting DNA damage most probably inhibited DNA replication and cell proliferation (Vock et al. 1998; Kirkland and Muller 2000). On the other hand, the high frequency of chromosome aberrations may have triggered mitotic selection and pressured cell proliferation (Madle et al. 1993; Vock et al. 1998; Galloway et al. 1998; Hillard et al. 1998; Armstrong et al. 1992; Kirkland and Muller 2000; Kocaman et al. 2014). The cell death might have arisen from the oxidative stress and the high genotoxic effects that caused to apoptosis and necrosis which is the main mechanism of the cytotoxicity (Bakkali et al. 2008; Kocaman et al. 2011).

Copper (Cu) is essential for many biological systems as a cofactor of enzymes including cytochrome C oxidase, tyrosinase, dopamine-β-hydroxylase, superoxide dismutase (Suzuki et al. 2002; El-Gendy et al. 2009) and it has a very important role in hemoglobin synthesis involved in Fe metabolisms (WHO 1974). The estimated daily requirement of copper is 2 mg for adults because the copper intoxication may cause to hemolytic anemia (WHO 1974). On the contrary, the exceeding intake amount of Cu accumulated in liver and other organs and caused several diseases in humans such as chronic renal and liver disfunctions (Gunay et al. 2006; Hloch and Charvát 2012). The same results were reported for animals (Hurwitz et al. 2014; Ciji and Bijoy Nandan 2014). Woimant and Trocello (2014) divided the Cu disorders into two classes, in which one of them was genetically and related to inherited Cu transport disorders. The other ones are Cu disorders, which are associated with Cu deficiency or exceed. The mutant ATP7B gene caused Cu accumulation in tissue resulting in clinical disorders known as Wilson disease (Grazyna et al. 2014). The high concentrations of Cu led to oxidative stress and toxicity in all living beings (Gupta et al. 1996; Giller et al. 1998; Srivastava et al. 2006; Ciji and Bijoy Nandan 2014).

Even though our study on copper oxychloride and copper oxide nanoparticles demonstrate genotoxic and cytotoxic effects, copper oxychloride is still widely used as a fungicide. About twenty thousand metric tons of copper oxychloride were used in Turkey alone in 2013 (https://www.zauba.com). The most serious risk is that the copper oxychloride is still not classified as a mutagen or potential carcinogen for humans. Our short-term in vitro exposure has clearly shown the genotoxic effect of copper oxychloride, but for a better understanding of its genotoxicity long-term carcinogenic studies and in vivo animal genotoxicity studies are needed.

Conclusion

Our in vitro study has confirmed that copper oxychloride is genotoxic and cytotoxic to human lymphocytes. Therefore, it represents a certain risk for the environment and humans, and future studies should investigate that risk, especially via the food chain. In the meanwhile, caution is advised when using copper oxychloride as a fungicide for the sake of our and the next generations’ health.