Liver histopathology of Baltic grey seals ( Halichoerus grypus ) over three decades

The liver plays an important role in the metabolism and elimination of endogenic and exogenic lipid-soluble compounds. Multiple studies have shown that polychlorinated biphenyls (PCB) and dichlorodiphenyl tri-chloroethane (DDT) lead to morphological changes in liver cells. The aim of the present study was therefore to analyse liver changes over time in Baltic grey seals ( Halichoerus grypus ) and to correlate these with historical PCB and DDT contaminations. A total of 191 liver samples were collected between 1981 and 2015 in the Gulf of Bothnia and northern Baltic Proper. Six histological features were evaluated, including portal mononuclear cell infiltration, random mononuclear cell infiltration, lipid granulomas, hepatocellular fat vacuoles, hepatic stellate cells and mild multifocal bile duct hyperplasia accompanied by portal fibrosis. Three of the six lesions showed a significant correlation with age. Furthermore, a positive correlation between portal mononuclear cell infiltration and mild multifocal bile duct hyperplasia was found. Additionally, lipid granulomas were significantly correlated with hepatic stellate cells. More importantly, hepatic stellate cells and mild multifocal bile duct hyperplasia were correlated with adipose tissue (blubber) concentrations of Ʃ PCB, measured in a subsample (n = 34) of all individuals. No correlation with lesions and Ʃ DDT concentrations were found. These results show that age is an important factor for the development of these liver lesions, but PCBs burden may be an influencing factor. This is in agreement with previous studies of marine mammals in the Baltic Sea as well as in the Arctic. We therefore conclude that not only age of the animals, but also exposure to PCBs should be taken into account when understanding and evaluating the current health status of Baltic grey seals.


Introduction
In the 1970s, a marked decrease in the population number of Baltic grey seals (Halichoerus grypus) was observed (Helle 1980).This was probably associated with bioaccumulation of organochlorines (OC) such as PCBs and DDTs (Desforges et al., 2016, Helle 1980).The main effect causing the population decrease was the loss of reproduction capacity of female grey seals, as PCBs and DDTs lead to uterine occlusions or stenosis and tumours (leiomyoma) in the uterine wall (Bäcklin et al., 2003;Helle 1980Helle , 1983;;Reijnders 1986).Furthermore, PCBs and DDTs are known to affect the bones, teeth, adrenal glands, liver and kidney tissue of Baltic seals (Bergman et al., 2001;Lind et al., 2003;Schmidt et al., 2020).PCBs and DDTs are organochlorines, which are the most widely used chemicals over the past seven-eight decades.Multiple publications report on organochlorines and their effects on the immune, endocrine, reproduction and skeletal systems of marine mammals (Beineke et al., 2005;Bergman 1999;Butt et al., 2007;Kimbrough et al., 1971;Routti et al., 2008).
In marine mammals, such as ringed seals, polar bears (Ursus maritimus) and North Atlantic long-finned pilot whales (Globicephala melas), PCBs and DDT lead to liver changes, especially fat globules, necrosis or mononuclear cell infiltrations, and also to renal lesions found in glomeruli and tubules (Bergman et al., 2001, Sonne et al., 2005, 2007b, 2010).Sonne et al. (2007aSonne et al. ( , 2007b) ) examined the effect of mercury and contaminants on Greenland polar bears.The results from these studies showed that high mercury concentrations accumulated in the liver tissue and were in the range of adverse toxic levels.Their studies also found multiple different changes in the liver, which cannot be ascribed to specific etiological factors.Similar results were found in North Atlantic long-finned pilot whales, where also a relationship between mercury and organochlorines in the development of liver lesions existed (Sonne et al., 2010).In female grey and ringed seals from the Baltic Sea, a significant positive correlation between DDT and PCB concentrations and the two types of renal changes, CWT (glomerular capillary wall thickening) and CWN (glomerular capillary wall nodules) were found (Bergman et al., 2001).Sledge dogs (Canis familiaris) in Greenland showed various renal lesions such as diffuse thickening of the glomerular capillary wall ascribed to OC exposure and recurrent infections caused by different bacteria, viruses and parasites, age-related changes or a combination of these (Sonne et al., 2007b).Similar lesions were also found in other domestic animals (Maxie, 2012) and humans (Cotran et al., 1999).
Industrial chemicals, OCs among these, are known to affect grey seals' overall health status including immune and reproductive systems due to their position as top predators (ICES, 2003).The species is essential for the native ecosystem due to its longevity and is supposed to have a significant effect on structuring its ecosystem (Apollonio, 2002;Ray, 1981).For that reason, grey seals have been used as a sentinel species in the monitoring of the overall health and status of the ecosystem (Gulland and Hall 2005).After the decrease in numbers in 1970, the grey seal population recovered very well in the Baltic region (Roos et al., 2012).Grey seals are an essential component of the ecosystem and have been heavily affected by PCBs and DDTs in the past.A positive sign is the current recovery of the population.However, no exact findings about the health status of the grey seals in the Baltic Sea exist.The aim of the present study was to determine the health status of the seals by documenting changes in livers and lesion patterns, which has not yet been done for the examined timespan.A further aim thereof was to investigate whether a link between pollutants and aforementioned changes exists or whether other factors have an impact on the liver.As no reports on liver lesions in Baltic grey seals have been published so far, liver tissues from 191 Baltic grey seals from the period 1981 to 2015 were histologically examined to detect temporal changes and relationships to OC exposure.

Samples
All liver samples (n = 191) were collected from animals found from 1981 to 2015 in the Gulf of Bothnia and in the northern Baltic Proper in the Baltic Sea (Fig. 1) and were provided by the Swedish Museum of Natural History (SMNH) in Stockholm.The animals were collected and necropsied within a long-term environmental monitoring project funded by the Swedish Environmental Protection Agency.The samples used for the study originated from grey seals, which had died either through bycatch, stranding or had been shot during the hunt along the Swedish coast.The animals had been shot in accordance with the existing Swedish hunting law.The samples were collected from animals of varying degrees of autolysis but none of the animals had been frozen Fig. 1.The shaded area from which grey seal liver samples were obtained.The samples were collected along the Swedish coast of the Gulf of Bothnia as well as in the Baltic Proper along the Swedish southern and eastern coast.before sampling.Necropsies and carcass collection were conducted as described by Bergman (2007) and liver samples were fixed in 4% buffered formaldehyde and embedded in paraffin wax until further investigations.

Age determination
The age of 191 animals was determined by the growth layers counted in the cementum of the canine teeth collected from the grey seals.Untreated thin sections were cut with a circular diamond saw as described by Lockyer et al. (2010) and counting of the cementum layers was performed by two technicians at the SMNH.The grey seals were categorised into three age classes: adult males (≥8 years old), adult females (≥6 years old), subadult males (4 ≥ 7 years), subadult females (3 ≥ 5 years) and juveniles (remaining individuals) (Atkinson 1997).
The liver samples consisted of 104 juveniles (38 females (≤2 years) and 66 males (≤3 years)), 35 subadults (14 females and 21 males), 23 adult females (6 ≥ 41 years) and 29 adult males (8 ≥ 33 years).Out of all samples, eight grey seals had been found during the breeding season (February to March), 32 individuals in the moulting season (April to May), 139 grey seals during the summer and autumn (June to November) and 12 grey seals during the winter season (December to January).

Histology
The 5 µm thick paraffin sections were stained with haematoxylineosin (HE).
Six histological features were evaluated and classified as follows: 1

PCB and DDT analyses
To investigate the effect of PCB and DDT on liver changes, the contaminant concentrations of 34 of the examined individuals from this study were used (Tables S2, S3, Supplementary).Ten PCB congeners (28, 52, 101, 105, 118, 138 + 163, 153, 156, 180) as well as three DDT congeners (p,p'-DDE, p,p'-DDD, p,p'-DDT) were examined.Before 1992, total PCB and ƩDDT had been analysed by gas chromatography (GC) equipped with packed columns.After that, the contaminants were analysed by GC with capillary columns.For a detailed description of the analytical methods, see Jensen et al. (1983) and Eriksson et al. (1997).Detailed information about the extraction procedures can be found in Jensen et al. (1983).

Statistical analyses
The statistical analyses were performed with R (version 3.4.3;Ihaka and Gentleman 1996) using the packages "nlme" (Pinheiro et al., 2019) and "glmulti" (Calcagno and de Mazancourt, 2010).Level of significance was set at p ≤ 0.05.In the small data set on contaminants, a trend was also considered between 0.05 < p ≤ 0.10.
Generalised linear models (GLM) using a binomial distribution and logit as link function were used to test the effects of age, age group, sex, season and the sampling year for the presence or absence of each liver lesion (regardless of dissimilation).The function "GLMulti" was used, which automatically compares and ranks all possible models based on the Akaike information criterion (AIC).The most parsimonious model was selected based on AIC, and assumptions of the GLMs (normality, homogeneity, independence) were checked.To assess correlations between lesions, portal mononuclear cell infiltration (Lesion 1) was tested against mild multifocal bile duct hyperplasia accompanied by portal fibrosis (Lesion 6), as well as hepatic stellate cells (Lesion 5) being tested against lipid granulomas (Lesion 3).
The relationship between liver changes and PCB levels, as well as DDT levels, was additionally tested with a GLM.Thereafter, the relationship between PCB levels and sex was tested with a linear regression model (LM).The same was done for DDT levels and sex.

Results
Three of the six lesions showed significant differences among age (Table 3; Fig. 3).The prevalence of two lesions changed significantly during the study period.
Two types of cell infiltrations could be observed in the livers of the examined grey seals.These were random mononuclear (intralobular) cell infiltration (Lesion 2) and portal mononuclear cell infiltration (Lesion 1).Random mononuclear cell infiltration was found in 70% of the sampled animals and was the most frequently found infiltration type (Table 1).Nevertheless, no significant relationship with any of the variables (age, year or season) could be seen.With 46%, portal mononuclear cell infiltration was the second most common infiltration type in the liver and showed a slightly positive significant relationship with age (GLM, p = 0.028; Fig. 3; Table 2; Table 3).This lesion decreased during the study period, but the dissimilation increased (GLM, p = 0.035; Fig. 4; Table 2; Table 3).Furthermore, a positive relationship between portal mononuclear cell infiltration and mild multifocal bile duct hyperplasia accompanied by portal fibrosis could be seen (GLM, p < 0.001; Table 2; Table 3).
Hepatic stellate cells (Lesion 5) could be found in 35% of the samples and there was a negative relationship with age (GLM, p = 0.02; Fig. 3; Table 1; Table 2; Table 3).Besides, a significant relationship between hepatic stellate cells and lipid granulomas was found (p < 0.001).
Mild multifocal bile duct hyperplasia accompanied by portal fibrosis (Lesion 6) was found in 16% of the examined grey seals (Table 1).The difference between mild multifocal bile duct hyperplasia accompanied by portal fibrosis and age was slightly positively significant (GLM, p < Fig. 3.The probability of portal mononuclear cell infiltration (Lesion 1) and mild multifocal bile duct hyperplasia (Lesion 6) increased significantly with age, whereas hepatic stellate cells (Lesion 5) decreased significantly with age.The y-axis shows the probability of each liver lesion (absence: 0; presence: 1), while the x-axis shows the age and the red line shows the changing probability with respect to age.There is a positive correlation for the portal mononuclear cell infiltration (Lesion 1) and mild multifocal bile duct hyperplasia (Lesion 6), but not for hepatic stellate cells (Lesion 5) (Table 1).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) B. Schmidt et al. 0.001; Fig. 3; Table 2; Table 3).Furthermore, a significant relationship between mild multifocal bile duct hyperplasia and portal mononuclear cell infiltration was found (p < 0.001).Only two grey seals (one female and one male) showed hepatocellular fat vacuoles (Lesion 4).

Liver changes vs. contaminants
Liver changes were analysed against PCB and DDT concentrations for 34 of the examined individuals sampled in the Gulf of Bothnia and northern Baltic Proper.This study showed that the presence of hepatic stellate cells (Lesion 5) and PCB concentrations in blubber negatively correlated, as less hepatic stellate cells could be found with increasing PCB concentrations (GLM, p = 0.063).In contrast to that, the correlation between the presence of mild multifocal bile duct hyperplasia accompanied by portal fibrosis (Lesion 6) and PCB was positive, as more mild multifocal bile duct hyperplasia could be found with increasing PCB concentrations (GLM, p = 0.79).No relationship was found between the liver changes and DDTs.Concentrations of both PCBs and DDT showed a decrease during the study period (GLM, p PCB = 0.054; p DDT = 0.027, Fig. 5).

Discussion
The Baltic grey seals underwent significant population declines during the 1960s-1970s due to exposure to persistent organic pollutants including PCBs and DDTs (Desforges et al., 2016, Helle 1980).The main reasons were obstructions of the sexual organs of females including uterine occlusions and uterine tumours, these leading to sterility (Helle 1980(Helle , 1983;;Reijnders 1986).Many of the seals affected by the Baltic Seal Disease Complex (BSDC) between 1960 and 1970 had adrenal cortical hyperplasia, probably related to extreme physical stress, which led to an increased production of cortisol (see review by Sonne et al., 2020aSonne et al., , 2020b)).Most neoplastic changes were found in older individuals, 20-40 years of age, with a prevalence of 65%.Furthermore, other pathological changes were reported, such as renal glomerulopathy (changes in glomerular structure and function of the kidneys), skull and claw lesions, interstitial nephritis as well as arteriosclerosis and intestinal ulcers (Bergman andOlsson, 1985, Bergman, 1999).
However, no reports on liver lesions in Baltic seals have been found in the literature so far.The present study showed that the described liver lesions are not indicative of a particular known cause.They may be the result of age-associated exposure to infectious agents or environmental toxic chemicals, e.g.PCBs or mercury, or a combination of both (Sonne et al., 2005(Sonne et al., , 2010(Sonne et al., , 2013)).Mononuclear cell infiltration was the most frequently encountered lesion.This may be caused by hepatic or systemic infections of microorganisms as well as exposure to cytotoxic chemical compounds (Kelly 1993, Sonne et al., 2005).
Significant relationships between lipid granulomas and hepatic stellate cells indicate that the fasting and feeding periods, which are linked to vitamin A detoxification, may play a role in this relationship (Sonne et al., 2005).Grey seals generally fast during the breeding and moulting season, and during the rest of the year (June and onwards)   they consume fatty prey to regain good body conditions (Breed et al., 2009, Scharff-Olsen et al., 2019).When grey seals feed on predominantly fatty prey over a longer period of time, this may lead to an increased development of hepatic stellate cells and lipid granulomas and is probably a naturally occurring phenomenon linked to vitamin A detoxification (Sonne et al., 2005).In grey seals, hepatic stellate cells store fat and vitamin A. However, this is compromised by liver damage, which then ultimately may affect function and overall health, possibly resulting in hepatic fibrosis (Leighton et al., 1988, Senoo et al., 2001).The level of vitamin A is associated with a diet rich in vitamin A that is stored in specialised hepatic stellate cells for means of detoxification (Sonne et al., 2005).The reason for the negative relationship between age and hepatic stellate cells found in the present study is unknown.Nevertheless, it has previously been observed in polar bears where it is most likely related to the end of weaning and the subsequent dietary changes (Derocher and Stirling, 1994).
The mild multifocal bile duct hyperplasia which is accompanied by portal fibrosis has previously been reported in other wildlife species such as polar bears and beluga whales (Delphinapterus leucas) as well as Greenland sled dogs (Sonne et al., 2005, 2007a, 2008, Woshner et al., 2002).In the present study, it was associated with age similar to that in findings of other species including bears and different cetacean species.(Kelly 1993;Prunescu et al., 2003;Sonne et al., 2005;Woshner et al., 2002).Bile duct proliferations were also reported in mammals exposed to mercury and PCB exposure, including captive controlled studies of mink (Bergman et al., 1992;Kelly 1993;Sonne et al., 2005;Sonne et al., 2013;Sonne et al., 2018).The positive relationship between mild multifocal bile duct hyperplasia and portal mononuclear cell infiltration found in the present study could not be found in previous studies of other marine mammals (Sonne et al., 2005(Sonne et al., , 2008, and the reason for the relationship is unknown.

Temporal trends
In the present study, livers from animals over 34 years of age were examined, within a timeframe in which the grey seals were exposed to PCB and DDT.Only two of the six lesions showed a correlation during the studied period.The presence of lipid granulomas (Lesion 3) increased over the years (Fig. 4).A wide range of disorders can explain such an increase in lipid granulomas (Culver et al., 2016;Cullen and Stalker, 2016).They are a response to hepatocellular injury with an increasing susceptibility to injury induced by exposure to cytokines or endotoxin.Fatty livers are more likely to suffer damage from a wide range of toxic and nutritional insults (Cullen and Stalker, 2016).In order to ascertain which disorders explain the presence of lipid granulomas (Lesion 3), further analyses of the individuals need to be performed (e.g. more contaminant analyses or food analyses).The presence of portal mononuclear cell infiltration (Lesion 1) decreased over the years (Fig. 4).This can be a combination of different infections as well as PCBs.

Contaminant exposure
According to Sonne et al. (2020a), the blubber threshold concentrations in seals in which the disease complex arose was around 100 μg/ g lw ( ∑ PCB17 range: 27-390 μg/g lw; DDT [p,p'-DDE, p,p'-DDD and p,p'-DDT] range: 12-970 μg/g lw), being among the highest ever measured in wild phocids (Table 2) (Jensen et al., 1979;Letcher et al., 2010;Roos et al., 2012).The concentrations of these seals in the present study ranged from 8.5 to 1848 mg/kg lw for PCBs and 2.3-61.1 mg/kg lw for DDT.These are above the stated thresholds and therefore it cannot be excluded that the present pathological findings are to some extent ascribed to contaminant exposure as well as age and re-current infections being the main factors.
Liver histology in relation to environmental levels of PCBs and DDTs has previously been reported for marine and terrestrial mammals (Hori et al., 1982, Prunescu et al., 2003, Sonne et al., 2005, 2007b, 2008).In the present study, the visibility of hepatic stellate cells showed a positive trend with PCB concentrations, whereas mild multifocal bile duct hyperplasia accompanied by portal fibrosis showed a negative trend with PCB.In terrestrial animals, like Cynomolgus monkeys (Macaca fascicularis), bile duct proliferation has also been associated with PCB exposure (Hori et al., 1982).In Arctic animals, this lesion was found in animals with higher concentrations of organohalogen contaminants (Sonne et al., 2005(Sonne et al., , 2008)).These results can give an insight into the possible effects of PCBs on the liver.They also indicate that animals exposed to a higher contaminant concentration may show significant results in the relationships between contaminants and liver lesions.
In the present study, a decrease in the correlation between the prevalence of portal mononuclear cell infiltration (Lesion 1) and collection year was found.In the same period, contaminant concentrations of PCBs and DDT likewise decreased, this suggesting that the decrease may be linked to the decrease in contaminant loads.

Conclusions
The study showed high prevalence of liver lesions in Baltic grey seals.The liver lesions can be related to age and in the case of Hepatic stellate cells and mild multifocal bile duct hyperplasia also to PCB contamination.These may be important factors in pathology development together with bacterial and viral infections.

Fig. 5 .
Fig. 5.The distribution of PCB and DDT relating to the examined timeframe (1981-2015).Both contaminants decreased with time.The y-axis shows ƩPCB and ƩDDT, while the x-axis shows the year.

Table 1
Prevalence and distribution of lesions and lesion grades in 191 grey seals from the Gulf of Bothnia in the period from 1981− 2015.

Table 2
Most parsimonious generalised linear model structures and corresponding summary statistics to investigate the impact of different parameters on liver lesions in 191 grey seal livers.

Table 3
Results of the most parsimonious generalised linear model.Three variable (age, season and year) are used; breeding is given as reference level for season and significant terms are indicated in bold.