E171 TiO 2

In the present research, the same stock suspension of E171 TiO 2 from our previous experiment17 was used. Approximately 30% of the particles in suspension are <100 nm in diameter. Before its use, the stock suspension was tested for bacterial and fungal contamination by means of inoculum on standard bacterial/fungal media. The stock suspension was confirmed to be sterile as no microorganism grew in the Petri dishes. The E171 (C.I. 77891) was manufactured by a commercial supplier - Fiorio Colori of Italy. The same stock suspension was used in order to provide a more reliable comparison between the previous and present experiments. Previously, we provided a comprehensive physico-chemical characterization of this particular batch of E17155. In summary, E171 is at least 99% pure, and has an anatase crystalline structure. The specific surface area of E171 is 6.137 m2 g−1, the pore volume is 0.123 cc g−1, and the pore diameter is 2.97 nm. It has a Ti 2 p3/2 peak at 463.8 eV and 2p1/2 peak at 458.0 eV, while a small shoulder at 532.5 eV implies that the surface is partially covered with hydroxide groups. The particles are amorphous-spherical-like with sharp and well-defined clean edges. The mean particle size ± standard error of the mean (SEM) is 167 ± 50 nm.

D. melanogaster care and housing

The fruit flies, Oregon-R-C (Stock number 5) wild-type strain (Bloomington Drosophila Stock center at Indiana University, USA) were housed en masse at an optimum density as recommended by Bloomington Drosophila Stock Center, Indiana, USA. They were maintained at optimal conditions of 25 ± 1 °C, a 12/12-hour day/night regime and 60% humidity. A standard cornmeal-based feeding medium was used, consisting of 10% cornmeal, 9% sugar, 2% yeast and 2% agar with the addition of the fungicide Nipagin (2.50 mg mL−1) (Alfa Aesar GmbH & Co KG, Germany) diluted in 96% EtOH.

Exposure concentration

The diet of each experimental group contained 0.014 mg mL−1 of E171 in the feeding medium, in addition to the above-mentioned ingredients, and the E171 was homogenously mixed into it. Flies were not on a nutrient restriction diet and therefore addition of E171 could not interfere with the calories intake. Briefly, before adding the E171 suspension to the liquid feeding medium, the stock suspension was sonicated for 2 min in a standard Fisherbrand sonicator bath to disperse the particles. Then, the E171 stock suspension was added to the warm, freshly prepared liquid feeding medium and stirred to homogenize. The liquid feeding medium was poured into 50 mL vials to cool down, and yeast extract was added to the surface. A concentration of 0.014 mg mL−1 E171 in the feeding medium was chosen in order to provide an average exposure of 20 mg kg−1 larvae/adult fly bw per day. An exposure concentration of 20 mg kg−1 larvae/adult fly bw per day was chosen, as it is believed to be close to the maximum possible exposure scenario in humans. For the maximum level exposure assessment scenario, the mean exposure estimates ranged from 0.4 mg/kg bw per day for infants and the elderly to 10.4 mg/kg bw per day for children, while at the 95th percentile, exposure estimates ranged from 1.2 mg/kg bw per day for the elderly to 32.4 mg/kg bw per day for children7. An adult D. melanogaster weighs on average 0.8 mg56 and eats 1.5 µL of feeding medium per day57,58, which is 1.7 times its body mass. A larva has an average mass of 1.8 mg59, and can eat about 3 µL of feeding medium per day assuming it also eats 1.7 X its body mass per day. This means that in order for an adult fly to be exposed to an E171 concentration of 20 mg kg−1 bw per day the amount of E171 in the feeding medium should be 0.016 mg mL−1. For the same level of larvae exposure, it should be 0.012 mg mL−1. In practice, it is not very plausible to prepare and maintain large numbers of different feeding mediums for 20 generation of flies and since the two concentrations differ by only 30%, all feeding media were prepared with an average concentration of 0.014 mg mL−1. Therefore, the adults were effectively exposed to 17 mg kg−1 bw per day of E171 and the larvae to 23 mg kg−1 bw per day of E171. This is also in line with the current understanding that children are exposed to higher levels of E171 than adults7.

Experimental setup

The experiment included one parental generation (F 0 ) and 20 filial generations (F 1–20 ) of D. melanogaster. The F 0 was chosen randomly from the en masse population and consisted of young adults, approximately 6 days old. These adults were randomly divided into 2 groups. Each group consisted of approximately 50 males and 50 females, which were housed separately. One group served as a negative control, while the second group received E171 in the feeding medium. From this point onward, these two lines were never crossed until F 20 , and adults from the E171 or control groups mated separately. The F 0 cohort was allowed to feed for 3 days, after which 10 males/females from each group were paired randomly. Each pair was kept separately in a 50 mL tube. The F 1 generation was formed from the first batch of eggs from each pair. The F 0 adults were removed from the tubes within 8 h of laying their eggs, while the eggs remained in the tubes and were incubated until the larvae hatched. Within the first 24 h of incubation time, all of the eggs were counted before hatching. Incubation was continued until the larvae reached the pupa stadium, then all of the pupae were counted. Immediately after eclosion, the adults were counted, separated by gender and transferred to new 50 mL tubes with E171 or control feeding medium in order to prevent uncontrolled mating. Once the adults reached approximately 6-days–post eclosion, 10 random male/female pairs per control or treatment were paired and each pair was transferred to a new 50 mL tube with fresh medium. The paired males and females always originated from a different parent in order to avoid inbreeding and a bottleneck effect. The pairs mated for 20 days (actual post eclosion age in days 6–25), and each day adults were transferred to a new medium, while the eggs were counted and observed every day until eclosion. The developmental time - DT (larva to pupa; larva to adult) was calculated for each of the 10 replicas for each day according to the following formula

$$DT=\sum _{d=1}^{x}\,{n}_{d}\,\ast \,d/{n}_{t}$$

where n d is the number of pupating larvae/emerging flies d days after the eggs were laid, and n t is the total number of individuals pupating/emerging at the end of single generation experiment. In some cases, a female would lay unfertilized eggs or it would lay too few eggs. Additionally, some females would stop laying eggs completely after a certain day. If a batch of eggs produced <10 larvae/adults at the end of the cycle it would be excluded from calculation of the DT, as the low number of pupating/emerging individuals would potentially skew the results. The F 2 cohort was formed from the eggs of the F 1 generation that were laid on the 1st day (actual age of parents was 6-days-post-eclosion), as D. melanogaster is then at its full reproductive maturity60. The same procedure was repeated until F 20 . A pair of flies would occasionally not produce eggs on the 1st day, and in that case the first eggs that were produced on the next days were counted and used for creation of the next F generation.

Due to the high number of mating pairs, and the number of eggs, transfers to new mediums, etc., the daily DT was calculated only for generations F 1 , F 10 , and F 20 . Fecundity and egg to adult viability was calculated for each generation. In addition, these were calculated for each of 20 consecutive days of eggs laid in generations F 1 /F 10 /F 20 . Sometimes, in generations F 1 /F 10 /F 20 , the male or female would die before the 20 days of laying eggs had finished. If that was the case, the missing mate was not replaced and data were recorded only until the last day that both the male and female were alive.

The F 20 generation had a slightly different experimental design. In addition to the standard setup used in the F 1 and F 10 generations, F 20 contained two extra treatment groups that consisted of control males crossed with E171 females; and E171 males crossed with control females. The aim of the crossing was to determine if a long multigenerational exposure to E171 resulted in a gender-specific reproductive/developmental effects. Due to the crossings, and therefore the larger number of treatment groups, the number of replicas for F 20 had to be reduced to 5–7 per group.

Genotoxicity

The genotoxicity of E171 TiO 2 was evaluated in vivo in the anterior midgut of D. melanogaster using the alkaline version of the comet assay. Phosphate-buffered saline (PBS) without calcium and magnesium, agarose for DNA electrophoresis, low-melting point agarose (LMA), and collagenase were obtained from Alfatrade Enterprise D.O.O.; methyl 4-hydroxybenzoate and ethyl methanesulphonate (EMS) were purchased from Sigma-Aldrich, St. Louis, MO, USA. In order to determine the potential for E171 to damage DNA in somatic cells across generations, the larvae of D. melanogaster were fed with the medium containing 0.014 mg mL−1 of E171, as previously described. EMS (1 mM in PBS) was used as a positive control. Genotoxicity was evaluated in generations F 1 , F 10 , and F 20 . The comet assay was performed as previously described61, with minor modifications62. Immediately before use, slides were stained with 80 µL of ethidium bromide (20 µg mL−1). The images were visualized and captured with the 40x objective lens of a Nikon fluorescence microscope (Ti-Eclipse) attached to a CCD camera. One hundred randomly selected cells (50 cells per two replica slides) were analyzed per treatment. The comets were analyzed by means of a visual scoring method63 and the total comet score was calculated according to64. The results were expressed as mean ± SEM and a statistical evaluation of the data was carried out by means of one-way analysis (ANOVA) using the SPSS statistical software package, version 13.0 for Windows. The significance level was set at p < 0.05.

Confirmation of E171 ingestion

The flies were housed and fed as described above. Half of the larvae per treatment or control group were collected for inductively coupled plasma mass spectrometry (ICP-MS) shortly before turning into pupae, while the other half were allowed to develop into adults. Emerging adults were fed for an additional 6 days before collection for ICP-MS. One gram of larvae and 1 g of adults were collected from the control or E171 groups per replica. A total of 5 replicas were included. The samples were weighed and preserved in 70% ethanol until further ICP-MS analysis. The samples were digested and processed in the same way as in our previous study65. Samples from the present study were processed in parallel with samples from our previous study65, and therefore shared the same recovery for titanium of 94.7 ± 1.1% (N = 3) from standard reference material (mussel tissue, SRM 2976, NIST) as we reported previously. Furthermore, standard reference material (oyster tissue, SRM 1566b, NIST, Gaithersburg, Maryland, ZDA) containing 12.24 + 0.39 mg/kg of Ti was digested and analyzed in the same way as the samples. The Ti concentration in the digested SRM 1566b was determined to be 12.25 + 0.44 mg/kg (N = 3). The E171 intake by the larvae or adults was calculated using the following equation: E171 conc. = (Ti conc. of the experimental sample − background Ti conc. of the control) × mass ratio of E171/Ti. The mass ratio of E171/Ti = 1.6684.

Histopathology

Adult flies and larvae from the F 1 and F 19 generations were euthanized in ethanol, fixed in 10% neutral buffered formalin, processed routinely into paraffin blocks, cut in step sections at 5 microns, stained with hematoxylin and eosin, and examined microscopically under bright-field conditions. At least 10 sections from 10 adults and 10 larvae were examined microscopically. Multiple organ systems of the adult were evaluated, including the exoskeleton, eye, central nervous system, mouthparts, salivary gland, foregut, midgut, hindgut, skeletal system, reproductive system, and fat bodies. Organ systems of the larvae evaluated include the exoskeleton, central nervous system, mouthparts, salivary gland, intestine, skeletal muscle and fat bodies. Tissue and cytomorphologic changes were recorded using a semi-quantitative severity scale from 0 to 5 where 0 = no lesion and 5 = severe lesion. Changes in the morphology of the abdominal fat body of adult flies was scored using the following criteria: 0 = >40% of fat body trophocyte volume is protein globules, 1 = 20–40% of fat body trophocyte volume is protein globules, 2 = 5–20% of fat body trophocyte volume is protein globules, 3 = rare intracytoplasmic protein globules, and 4 = no protein globules in fat body trophocyte cytoplasm.

Statistics

Unless otherwise noted, Repeated factorial Analysis of Variance (RANOVA) was used to check the effects of the treatment, generation number, and treatment * generation number interaction effects using Statistica 12.0 software. Before analyses, the data were analyzed using the Kolmogorov Smirnov and Lilefors tests for normality. If assumption of normality was not met, data were Box-Cox transformed before running ANOVA. Student’s t-test was used to compare the ICP-MS data. The comet assay was analyzed with ANOVA using the SPSS statistical software package, version 13.0 for Windows. Histopathology scores were analyzed by ANOVA and Tukey’s multiple comparison tests using GraphPad Prism 7.04. Only a p value of <0.05 was considered statistically significant.