Life span

Male C3B6F1 mice were supplemented beginning at 12 months of age with food containing krill oil or Lovaza. The dosages given and the rationale for them are discussed below. When the effects of the marine oils on longevity were analyzed together using the Mantel–Cox log-rank test, they significantly decreased life span by 6.6 % (P = 0.0321). Individually, Lovaza and krill oil non-significantly shortened mean life span by approximately 9.8 and 4.7 %, respectively (Fig. 1). These data probably were not significant individually because of sample size.

Fig. 1 Life span of mice fed a control diet or a diet supplemented with Lovaza or krill oil. Shown are the life spans of the control (filled circles), krill oil-treated mice (downward-pointing triangles), and Lovaza-treated mice (upward-pointing triangles). The percentage of mice remaining alive at the end of each time period shown is plotted. The controls began with 297 mice and the treatment groups with 36 mice each. The graph begins at 365 days of age, when treatments were started Full size image

These studies were a part of a larger study in which one control group of 297 mice and 58 treatment groups of 36 mice each were used to screen for the effects of different compounds on life span (e.g., Spindler et al. 2013a, b). Other of these treatments increased life span, although most had no effect. For example, treatment of the mice with β-adrenergic receptor blockers or 40 % caloric restriction (CR) produced a significant 8.4 and 23 % increase in life span (Spindler et al. 2013b). In contrast, other compounds produced no effect on life span (e.g., Spindler et al. 2013a, 2014). The sample sizes in this study are similar to those required for a Weibull survival analyses with a 75 % probability of detecting a 10 % increase in mean life span with a 1 % (β ≤ 0.01) probability of a false positive. The Weibull survival analysis is more stringent than the log-rank test (Jeske et al. 2014).

Rationale for dosages

Krill oil was administered at 1.17 g oil/kg diet, which is ~124 mg/kg body weight per day (mg/kg bw/day), or approximately 3 % of the oil in the diet. The calculation assumes a body weight of 39 g, which is approximately the median weight of the mice during the treatments (Fig. 2). Lovaza was administered at 4.40 g/kg diet, which is ~467 mg/kg bw/day, or approximately 11 % of the oil in the diet. An equivalent volume of soybean oil was removed from each diet to compensate calorically for the additions. The American Institute of Nutrition (AIN)-93 M diet contains 40 g soybean oil per kg diet, which provides only small amounts of EPA or DHA from the inefficient metabolic conversion of other fatty acids in the oil (Reeves et al. 1993; De Caterina 2011). The krill oil brand used is 23 % EPA and 6.5 % DHA by weight, while Lovaza is 55 % EPA and 45 % DHA by weight. We could not find a literature describing the use of Lovaza in mice. The recommended dose in humans is 4.0 g/day, which would provide 50 mg/kg bw/day for an 80-kg adult. The cross-species scaling factors used to adjust dosages between animals and humans suggest that mice should receive 8 to 12 times the effective human dosage of a drug to account for species-specific pharmacodynamic and pharmacokinetic differences (reviewed in Spindler 2012). The Lovaza dose used here is approximately 9.3 times the recommended human dosage per kilogram body weight. The krill oil dosage is modest in comparison to the dosages used in mouse studies to demonstrate a beneficial effect on serum and hepatic cholesterol and triglyceride levels (Vigerust et al. 2012; Tandy et al. 2009). For example, tumor necrosis factor alpha-transgenic mice fed a high-fat diet containing 5.8 % krill oil (approximately five times the dosage used here) had lower plasma levels of triacylglycerol and cholesterol and higher levels of hepatic mitochondrial and peroxisomal fatty acid β-oxidation and carnitine turnover (Vigerust et al. 2012). High fat-fed mice receiving krill oil at 12.5 g/kg diet (approximately 10 times that used here) had reduced hepatomegaly, hepatic steatosis, triacylglycerols, and cholesterol (Tandy et al. 2009).

Fig. 2 Body weights and food consumption of the mice. The symbols are as labeled in the figure. The notation Percent of Kcal consumed refers to the percent of the food eaten with respect to the amount presented to the mice in each treatment group per mouse during the preceding 30 days with respect to the amount consumed by the control mice during the same period. For the weights of the mice, the median weight plus or minus the standard deviation is shown. The differences between the weights of the groups were not significant, as judged using a linear mixed effects model as described previously (Spindler et al. 2013a) Full size image

Food consumption and body weight

To determine the possible effects of the compounds on food intake and body weight, the mice were fed a known number of calories each day, and both parameters were quantified throughout the study (Fig. 2). As is typical of meal-fed B6C3F1 mice, all the mice began to lose weight beginning at about 12 months of age, even though they were eating near ad libitum levels of calories (e.g., Spindler et al. 2013a, b; Martin-Montalvo et al. 2013). As in our previous studies, the mean weight estimates became increasingly unstable as days on diet increased, because the number of mice diminished with time (Spindler et al. 2013a, b). Thus, neither the Lovaza nor the krill oil diet produced a consistent treatment effect on body weight (Fig. 2). Anomalously, at 1,125 days of age, the Lovaza diet-fed mice consumed only 78 % of the food offered (Fig. 2). However, only five of the mice remained at this time. Three of these mice died in the ensuing month, and the remaining mice consumed a more “normal” amount of food.

Necropsies

Necropsy results are shown in Tables 1 and 2. The necropsied control mice were randomly chosen by age at death to approximate the age distribution of the entire cohort. Both Lovaza and krill oil consumption significantly increased the number of mice with peritoneal hemorrhagic diathesis (bleeding into the peritoneum; Table 1). The increase was 3.9-fold for Lovaza and 3.1-fold for krill oil. As mentioned in the “Introduction,” hemorrhagic diathesis is a postmarketing adverse reaction which has been reported for Lovaza by GlaxoSmithKline (2013). We were unable to determine the frequency or severity of this outcome from the information available. In our study, Lovaza treatment also produced a significant increase in the number of enlarged seminal vesicles (7.1-fold; Table 1). There was a 52 % reduction in the median liver tumor mass in the Lovaza-treated mice (Table 2). This size reduction may not have been the result of their shortened life spans, since a similar effect was not found for the krill oil-treated mice (e.g., Dhahbi et al. 2004; Spindler 2005). Lovaza and krill oil treatment produced a significant 4.1- and 8.2-fold increase, respectively, in the number of lung tumors (Table 1). Thus, marine oil consumption was associated with an increase in the onset, incidence, or severity of multiple adverse health outcomes.

Table 1 Necropsy results from the mouse longevity studies shown in Fig. 1 Full size table

Table 2 Liver tumor mass of the mice shown in Fig. 1 Full size table

Serum tests

To determine whether the decrease in life span was related to drug-induced toxicity, serum samples were analyzed from mice of the same sex and strain fed the Lovaza or krill oil diets (Tables 3 and 4). Most of the parameters measured were not changed significantly by the treatments, suggesting that the oils were not overtly toxic to the liver, muscle, kidney, or other organ systems. Lovaza produced a modest reduction in blood urea nitrogen (Table 3). However, this change was quantitatively modest, not found in the krill oil diet-treated group, and is therefore of uncertain significance. The Lovaza-treated mice did have unkempt hair coats, which can be a symptom of generalized illness.

Table 3 Serum tests for Lovaza diet-fed mice. Six treated and six control 19-month-old mice of the same sex and strain used in the life span studies were fed the Lovaza supplemented diet for 16 weeks prior to bleeding by heart puncture Full size table

Table 4 Serum tests for krill oil diet-fed mice. Six treated and six control 16-month-old mice of the same sex and strain used in the life span studies were fed the krill oil supplemented diet for 11 weeks prior to bleeding by heart puncture Full size table

The krill oil-treated group showed limited signs of toxicity. Their bilirubin, triglyceride, and glucose levels were modestly elevated (Table 4). Elevation of these parameters can be a sign of liver dysfunction. The Lovaza-treated mice also had numerically elevated triglycerides, although this change did not reach significance. High fat-fed mice receiving krill oil at 12.5 g/kg diet (approximately 10 times that used here) had reduced triacylglycerol and cholesterol levels (Tandy et al. 2009). However, we found an opposite effect on triglycerides for mice consuming the AIN-recommended levels of fat and carbohydrate (AIN-93 M diet; Table 3) (Reeves et al. 1993).