*9.2. Depression*

The effect of krill oil supplementation was evaluated on cognition and depression-like behaviors both in pre-clinical and clinical studies.

One of the first studies in this field was carried out for seven weeks in rats, which received krill oil 0.2 g/rat/day, or imipramine 20 mg/kg/day (used as an anti-depressant reference drug) or placebo. At the end of the treatment, the cognitive abilities were tested by the Aversive Light Stimulus Avoidance Test (ALSAT) while the potential anti-depressant effect was tested by the Unavoidable Aversive Light Stimulus (UALST) and the Forced Swimming Test (FST). The results showed that krill oil treated rats had a significant ability to discriminate between the active and the inactive levers in the ALSAT test since the first day of training. Moreover, rats treated with krill oil and impramine exhibited significant improvement in behavior features such as lower levels in the UALST test since day three, and shorter immobility time in the FST test. The investigation also involved the expression of brain-derived neurotrophic factor (Bdnf) which was found to be upregulated in the hippocampus of krill oil treated rats [146].

Similar results were obtained by Zadeh-Ardabili and colleagues on mice treated with fish oil, krill oil, vitamineB12, imipramine or saline 5 mL/kg once per day for 14 days starting after one week of the Chronic Unpredictable Stress (CUS) paradigm overnight procedure. During CUS procedures, light was used to stress the mice overnight with an illumination of 10W LED, 15 Hz for 12 hours for 3 weeks. The potential antidepressant effect of the treatments was tested by tail suspension test (TST) and FST. After animal sacrifice, the presence of oxidation markers was evaluated in the brain tissue. Both fish oil and krill oil significantly reduced the immobility factors and increased the time of climbing and swimming, similar to imipramine, when compared with the control group. Both the fish oil and the krill oil led to decreased malondialdehyde and hydrogen peroxide levels, decreased catalase activity, increased glutathione peroxidase levels and increased superoxide dismutase activities and glutathione levels in hippocampal tissue [147].

Another pre-clinical study, by Mendoza et al., investigated the role of krill oil on restraint stress in mice after reduced mobility. After two weeks of acclimation and handling, mice were immobilized for three weeks followed by a week dedicated to behavioral tests. During the four weeks of the study, the mice orally received PBS or cotinine (a nicotinederivative) at 5 mg/kg, or cotinine plus krill oil 143 mg/kg. The results showed that cotinine alone reduced both the loss in cerebral synaptic density, memory deficits, anxiety and depression-like behaviors, but that the co-administration of cotinine plus krill oil was more effective than cotinine alone in reducing depression-like behaviors linked with reduced mobility. This confirms krill oil plays a role in depression mechanisms [148].

These encouraging pre-clinical data encouraged van der Wurff and colleagues to design and carry out a year long randomized, controlled, double-blind clinical trial on the effects of a krill oil supplementation on adolescent behaviors linked to learning, cognition, visual processing and mental well-being. The study included 264 adolescents between the ages of 13 and 15 years, who were divided in two cohorts. Cohort I started with 400 mg/day of EPA + DHA or placebo, and after three months the dose was increased to 800 mg of EPA + DHA per day. Cohort II started with 800 mg of EPA+DHA per day. The effects of these treatments were assessed by Omega-3 Index finger-prick blood measurements, Centre for Epidemiologic Studies Depression Scale evaluation and the Rosenberg Self Esteem questionnaire. The authors concluded that there was no evidence of an effect of krill oil in reducing depressive feelings or in inducing higher self-esteem. However, the authors reported that the results were affected by low adherence and drop-out, and for these reasons they suggest caution when interpreting the data [149,150].

#### **10. Cancer**

Only a few studies carried out with respect to krill oil and cancer cell lines are available. In a screening study carried out on several cancer cell lines such as cells derived from histiocytic lymphoma (U937), leukemia (K562, HL60), human hepatocarcinoma (SMMC- 7721), bone metastasis of pancreatic cancer (PC3) and breast cancer (MDA-MB-231, MCF-7), a general inhibition of cancer cells proliferation by krill oil was observed [151].

In a comparison with fish oil, EPA and DHA incubated in human osteosarcoma cells for 24, 48 and 72 h, found only krill oil induced a significant inhibition of cancer cells proliferation (23, 50 and 64% of inhibition, respectively). Fish oil did not change the proliferation observed in control cells except for an increase observed after 24 h. On the contrary, EPA and DHA promoted cell proliferation and cell migration [152].

Some researchers have focused their attention on human colorectal cancer cells, in particular, Jayathilake and co-workers treated HCT-15, SW-480 and Caco-2 cells for 48 h with free fatty acid (FFA) extract from krill oil and fish oil. Their results indicated that krill oil and fish oil extracts inhibited cell proliferation in a similar manner but only an increase in mitochondrial membrane potential and consequent cell apoptosis was only observed with krill oil [153]. The same research group further investigated the antiproliferative effect of free fatty acid extract (FFAE) of krill oil on other human (DLD-1, HT-29 and LIM-2405) and murine (CT-26) colorectal cancer cells, in comparison with EPA, DHA (after 24 and 48 h) and oxaliplatin (after 24 h). Osteosarcoma, with colorectal cancer FFAE of krill oil, EPA and DHE inhibited cell proliferation and ROS formation in a similar way to oxaliplatin. FFAE of krill oil also induced a significant increase in caspase 3 and 9 levels which are markers of apoptosis [154]. An investigation of potential mechanisms of action was then carried out on DLD-1 and HT-29 cell lines treated with FFAE of krill oil at 8 and 24 h. From the evaluation of epidermal growth factor receptor (EGFR) signaling, the results indicated that FFAE of krill oil, at 0.03 and 0.12 μL/100 μL, induced reduction in EGFR, pEGFR, pERK1/2 and pAKT expression without any changes in total ERK1/2 and AKT levels. The expression of the ligand PD-L1 was significantly inhibited by FFAE of krill oil [155].
