Plastic marine debris and their additives: in vitro assessment of phthalates cytotoxicity and genotoxicity on European sea bass and bottlenose dolphin cell lines
Author(s)
Molino, Chiara
Date Issued
October 18, 2019
Type
Doctoral Thesis
Abstract
Marine pollution from anthropogenic litter has become a serious worldwide environmental concern,
resulting in multiple ecological consequences. Around 6.4 million tonnes of litter enter the oceans
each year, thus rendering not negligible litter impacts on the marine environment. Marine litter is
extensively distributed in the marine environment, and plastic debris accounts for the majority of litter
items in the sea (60 – 90 %), becoming a major source of pollutants. Among them, Di(2-ethylhexyl)-
phthalate (DEHP) is the most abundantly used plastic additive, resulting to be a potential ubiquitous
contaminant to the marine environment. DEHP and one of its primary metabolites, mono(2-
ethylhexyl) phthalate (MEHP), have been extensively studied in the last decades, demonstrating their
influence in biochemical processes both in humans and wildlife. However, studies on its toxicological
effects on marine organisms are still scarce, despite these studies could play a key role to solicit the
development of necessary measures to face this environmental emergency. In this PhD project, we
studied the cytotoxic, genotoxic, and mutagenic effects of DEHP and MEHP, by applying specific in
vitro tests, in two coastal species of ecological and economic value: the European sea bass embryonic
cell line (DLEC) and bottlenose dolphin skin cell line (TT); the latter was evaluated in comparison to
the standardized Chinese Hamster Ovary cell line (CHO).
DEHP results on DLEC showed a significant decrease in cell viability starting at 0.01 mM after 24 h
together with a significant increase in apoptosis and necrosis, morphological changes and cell
detachment. MEHP displayed a slight effect on DLEC viability but no effect in the induction of
apoptosis and necrosis at all tested concentrations. The higher toxicity of DEHP could be attributed
to a synergic effect of both DEHP and its metabolites, which are produced by the cellular metabolism
of the DEHP. Furthermore, DEHP caused a moderate increase in DNA strand breaks from 0.02 mM,
whereas MEHP did not enhance DNA fragmentation at the tested concentrations. Yet, both DEHP
and MEHP caused a dose-dependent increase of micronucleus frequency, which is considered a
marker of permanent DNA damage, displaying half of the micronuclei induced by MEHP, with
respect to DEHP, at the same concentration (10 µM). These outcomes suggested a possible aneugenic
effect of these compounds on DLEC cell line, probably mainly ascribed to MEHP. Our results
demonstrate that in vitro exposure to DEHP had a dose-dependent cytotoxic and genotoxic effects in
DLEC cell line, whereas MEHP resulted more dangerous than its precursor because it induces
genomic instability in the DLEC cell line without triggering cell death.
Results on the mammalian cell lines show a dose-dependent reduction in the viability of TT after 24
hours of treatment, starting from low/intermediate doses (0.02 and 0.1 mM of DEHP), and a slight
but not significant increase in necrotic cells, except for the 5 mM dose. Instead in CHO a significant
decrease in cell viability was observed, along with a significant increase in necrotic cells, at all tested
doses of DEHP. Although DEHP treatment of both lines did not increase DNA fragmentation at any
of the concentrations tested, a dose-dependent increase in the frequency of micronucleus was
observed, accompanied by a progressive decrease in cell proliferation. These data further support the
hypothesis that DEHP could have an aneugenic effect, probably due to the production of its
metabolites, in particular MEHP.
Furthermore, comparing the current results with those obtained in the study on the European sea
bass embryonic cell line, the degree of DEHP toxicity on the tree cell lines resulted in this decreasing
order: DLEC > CHO > TT.
In conclusion, the results of the whole work underline the importance of deepening the study of the
cytotoxic and genotoxic effects of phthalates on in vitro systems, especially for protected species, and
to undertake further investigations on their effects in in vivo and/or ex vivo cellular systems of marine
organisms.
Additional information
Dottorato di ricerca in Ecologia e gestione sostenibile delle risorse ambientali
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