### ELSEVIER Aquatic Toxicology 57 (2002) II-26



### Review 

# AQUATIC TOXICOlOGY

www.elsevier.com/locate/aquatox



# Role ofselenium toxicity and oxidative stress in aquatic birds

David J. Hoffman

# *

**~**
**~** USGS Patuxent Wildlife Research Center, 12011 Beech Forest Road, Laurel, lvJD 20708-4041, USA
Received 19 March 2000; received in revised form 18 July 2000; accepted 4 August 2001

Abstract

Adverse effects ofselenium (Se) in wild aquaticbirds have been documented as a consequence ofpollution ofthe aquatic environment by subsurface agricultural drainwater and other sources. These effects include mortality, impaired reproduction with teratogenesis, reduced growth, histopathological lesions and alterations in hepatic glutathione metabolism. A review is provided, relating adverse biological effects of Se in aquatic birds to altered glutathionemetabolism and oxidative stress. Laboratory studies, mainlywith an organic form ofSe, selenomethionine, have revealed oxidative stress in different stages ofthe mallard (Anasplatyrhynchos) life cycle. As dietary and tissue concentrations ofSe increase, increases in plasma and hepatic GSH peroxidase activities occur, followed by dose-dependentincreasesin theratio ofhepatic oxidized to reduced glutathione (GSSG:GSH) andultimatelyhepatic lipidperoxidation measured as anincreasein thiobarbituric acid reactive substances (TBARS). One ormoreofthese oxidative effects were associated with teratogenesis (4.6 ppmwet weight Se in eggs), reduced growth in ducklings (15 ppm Se in liver), diminished immune function (5 ppm Se in liver) and histopathological lesions (29 ppm Se in liver) in adults. Manifestations ofSe-related effects on glutathione metabolism were also apparentin field studies in seven species ofaquatic birds. Reduced growth and possibly immune function butincreased liver:body weight and hepatic GSSG:GSH ratios were apparent in american avocet (Recurvirostra americana) hatchlings from eggs containing 9 ppm Se. In black-necked stilts (Himantopus mexicanus), which contained somewhat lower Se concentrations, a decrease in hepatic GSH was apparent with few other effects. In adult American coots (Fulica americana), signs of Setoxicosisincludedemaciation, abnormalfeather loss andhistopathologicallesions. Meanliver concentrationsof28 ppmSe (ww) in the coots were associated with elevatedhepaticGSHperoxidase, depletion ofhepaticprotein bound thiols and total thiols, buta small increasein GSH. Diving ducks in the San Francisco Bay area exhibited apositive correlation between hepatic Se concentration and GSH peroxidase activity (r = 0.63, P<0.05), but a negative correlationbetween hepatic Se andGSHconcentration(r= - 0.740, P< 0.05). Inwillets (Catoptrophorussemipalmatus) from the San Diego area, positive correlations occurred between hepatic Se concentration and GSSG (r

### =0.70, P < 0.001), GSSG:GSH ratio, and TBARS. In emperor geese (Chen canagica) from western Alaska, blood levels of upto9.4ppmoccurred andwere associated withincreasedplasma GSHperoxidase activity (r

### =0.62, P<0.001), but withdecreasedplasmaGSSGreductaseactivity. WhenevaluatingSe toxicity, interactivenutritionalfactors, including otherelements and dietary protein, should also be taken into consideration. Further studies are needed to examine

*Tel.: 

### +1-301-497-5712; fax: 

### +1-301-497-5675. E-mail address: david_hoffman@usgs.gov (D.J. Hoffman).

0166-445Xj02f$

- see front matter. Published by Elsevier Science B.V. PII: S0166-445X(01)00263-612 D.J. Hoffman/ Aquatic Toxicology 57(2002) 11-26

therelationshipbetween differentforms ofenvironmentallyoccurring selenium, arsenic andmercury on reproduction, hepatotoxicity and immunefunction ofaquatic birds. Further selenium nutritional interaction studies may also help
to illucidate the mechanism ofselenium induced teratogenesis, by optimizing GSH and other antioxidant defense mechanismsin a mannerthatwould stabilize orraise the cell's threshold for susceptibility to toxic attackfrom excess selenium. It is concluded that Se-related manifestations ofoxidative stress may serve as useful bioindicators ofSe exposure and toxicity in wild aquatic birds. Published by Elsevier Science B.V.

Keywords: Selenium; Aquatic birds; Mallard (Anasplatyrhynchos); Glutathione; Oxidative stress; Hepatic

1. Introduction



### Agricultural drainwater, sewage sludge, fly ash from coal-firedpowerplants, andmining ofphosphates and metal ores contribute to the selenium (Se) burden in the aquatic environment (Eisler, 1985; 

### Heinz, 1996). 

### Historically, plant-derived Se in excess of4 ppm in the diet of chickens was found to impair reproductive success and to be teratogenic (Poley and Moxon, 1938). 

### In more recent years, adverse effects ofSe in wild aquatic birds have been documented as a consequence of pollution of the aquatic environment by subsurface agricultural drainwater and other sources. At the Kesterson National Wildlife Refuge, in the San Joaquin Valley ofCalifornia, mean Se concentrations of 73 

### mg/kg occurred during 1983 

### in submerged rooted aquatic plants that were consumed by ducks, and levels ofselenium averaged

over 100 

### mg Se/kg in aquatic insects (Ohlendorf, 1989). 

### Biological and physiological effects observed at Kesterson included avian mortality, impaired reproduction and teratogenesis, and histopathological lesions with alterations in hepatic glutathione (Ohlendorf et al., 1986, 1988; 

### Hoffman et al., 1988; 

### Ohlendorf and Skorupa, 1989). 

### Similar problems have been discovered· elsewhere in the western United States in at least

a dozen different locations (Skorupa, 1998) 

### but most notably in the Tulare Basin in California (Skorupa and Ohlendorf, 1991).

**~elenomethionine** 

### is a major form of Se in wheat (Olson et al., 1970), 

### soybean protein (Yasumoto et

### al., 1988), 

### and may be a majorform of

**s~** 

### in other plants

.eilstein an Whanger, 1987). 

### Th1s form of 

## Se is most likely an important 

# /t(exposure source for wild aquatic birds since the toxic thresholds ofthe form in eggs for decreased hatching success andteratogenicityfromlab studies with mallards are very close to those derived fromfield studies (Heinz, 1996). 

### Furthermore, the types and frequency ofmalformations caused in lab studies by selenomethionine are very similar to those seen in nature (Heinz et al., 1987, 1989; 

### Hoffman and Heinz, 1988). 

### Selenomethionine becomes particularly embryotoxic and teratogenic whenitexceeds 4ppmSeinthelaboratorydiet of mallards. At higher concentrations it becomes toxic to other stages of the mallard life cycle. Mallard ducklings exposed for 6weeks to dietary Se as selenomethionine suffered reduced survival and growth, with dose-dependent Se accumulation in the liver (Heinz et al., 1988) 

### and hepatotoxicity characterized by oxidative stress (Hoffman et al., 1989). 

### Other studies have quantified and described histopathological lesions of selenomethionine in adult mallards (Albers et al., 1996; 

### Green and Albers, 1997). 

### Dickson and Tappel (1969) 

### first proposed that Se toxicity is related to changes in intracellular concentrations ofreduced glutathione (GSH), and

excess Se has been shown to interactwith cellular sulfhydryls (Combs and Combs, 1986). 

### Most mammalian studies have examined effects ofexcess dietary Se in the selenite form. Ganther (1986) 

### demonstrated that Se at the oxidant state ofseleniteundergoes reductivemetabolism, utilizing GSH and NADPH as a source ofreducing equivalents. LeBoeuf et al. (1985) 

### reported that 3.0-6.0 

### ppm Se as sodium selenite in the diet of rats for 6 weeks caused a significant dose-dependent increase in hepatic GSH, in oxidized glutathione (GSSG), and in the GSSG:GSH ratio. When isolated hepatocytes were incubated with selenite (30-100 

### J.LM) in vitro, a depletion of thiolsincludingGSHwas observed (Anundiet al.,D.J. Hoffman/ Aquatic Toxicology 57(2002) 11-26 13 1984). Other studies revealed that selenite stimulates lipid peroxidation in vitro and in vivo (Dougherty and Hoekstra, 1982; Csallany et al., 1984). Spallholz (1994) concluded that Se toxicity manifests itself when oxidative damage exceeds antioxidant defenses or the ability of animals to form selenoproteins, selenoethers orelemental Se. Studies with mallards have compared Se as selenite and selenomethionine, butwith further focus
on selenomethionine because ofits likely biological role in nature. These studies have revealed oxidative stress in different stages ofthe mallard life cycle. Manifestations of the oxidative stress · caused by selenomethionine included alterations inhepaticglutathionemetabolismtowards amore oxidized state, accompaniedbylipid peroxidation (Hoffman and Heinz, 1988; Hoffman et al., 1989, 1991a). Inthese studies, as dietaryand subsequent tissue concentrations of Se were gradually increased, increases in plasma and hepatic GSH peroxidase activities occurred, followed by dosedependent increases in the ratio of hepatic oxidized glutathione (GSSG) to reduced glutathione (GSH), andultimatelyincreasedhepaticlipidperoxidation, measured as thiobarbituric acid reactive substances (TBARS). The purpose of this paper is to provide a review, interpretive discussion, and future direction for studies with aquaticbirds onbiological effects ofexcess Se as related to glutathione metabolism and oxidative stress. Studies measuring these end points include: (1) laboratorystudies, mainlywith mallards and (2) field studies with seven species of aquatic birds. While many ofthese investigations
were anintegrationoflaboratoryandfield efforts, with the latter related to aquatic birds mainly in California, USA, the responses and mechanisms
are ofinterest to aquatic toxicologists in general.

2. Laboratory studies and selenium-related oxidative stress in aquatic birds

2.1. Reproductive effects in mallards and black-crownednight herons
Findings from reproductive studies have indicated that Se as selenomethionine is considerably more teratogenic and generallymore embryotoxic than sodium selenite, probably due in part to much higher accumulation in eggs of Se when provided as selenomethionine in the diet than as sodium selenite (Table 1). When mallards were fed a control diet, diets containing 1, 5, 10, or25 ppm Se as sodium selenite, ora dietcontaining 10 ppmSeasseleno-D,L-methionine, Seat 10ppmas selenomethionine or 25 ppm as sodium selenite caused a 40-44%decrease in the total number of eggs that hatched compared with controls (Heinz et al., 1987; Hoffman and Heinz, 1988). Tenppm Seas selenomethioninewas moreteratogenicthan sodium selenite at 25 ppm. Selenomethionine (10 ppm Se) resulted in an incidence of13.1%malformations that were often multiple, whereas, sodiumselenite (10 and25 ppmSe) resulted in 3.6 and 4.2% malformations. The teratogenicity of selenomethionine was confirmed in a second experiment in which mallards received 1, 2, 4, 8, or 16 ppm Se as selenomethionine, resulting in 0.9, 0.5, 1.4, 6.8, and 67.9% malformations, respectively (Heinz et al., 1989). These malformations included hydrocephaly, microphthalmia, lower bill defects and foot defects with ectrodactyly. Bothforms ofSeincreasedtheincidence ofedema and stunted embryonic growth. Selenomethionine (10 ppm Se) resulted in a significant increase of approximately, 40% in plasma glutathione peroxidase activity and a 70%increasein sorbitol dehydrogenase activity (indicative ofhepatotoxicity)in hatchlings (Hoffman and Heinz, 1988). Sodium selenite (25 ppm Se) resulted in fourfold elevation in plasma uric acid concentration, indicative of renal alteration. Hatching success ofeggs laid by black-crowned night-herons (Nycticorax nycticorax) fed a 10 ppm Se diet, as seleno-D,L-methionine was not different from eggs laid by herons fed an unsupplemented diet (Smith et al., 1988). Developmental malformations commonly associated with Se exposure in other birds were not observed in heronembryos orhatchlingsinthe 10ppmgroup. However, 3-day-oldhatchlingsfromheronsfed 10 ppmSehad shorterradius-ulna andfemurlengths and higher hepatic TBARS (malondialdehyde) concentrations thancontrols. Se residues inheron eggs were lower than those reported in mallards**~**

Table I Laboratory studies on effects of excess selenium on glutathione metabolism and oxidative stress in aquatic birds

Species, age Form of dietary Se (concentration, Observation Effects References

ppm) period

Mallard, embryo sodium selenite (10) Through 0.5 ppm Se (ww) in eggs, decreased hatching Hoffman and Heinz, 1988

hatching success, caused edema and decreased growth,

increased

plasma GSH peroxidase activity

(25) 1.3 ppm Se (ww) in e:ggs, caused edema and

decreased

hatching success and growth, increased

plasma

GSH peroxidase activity and uric acid concentration b

Seleno-D,L-methionine (10) 4.6 ppm Se (ww) in eggs, caused malformations and **~**

decreased

hatching, increased plasma GSH **~**

peroxidase and SDH activities §

:!!

Black-crowned night Seleno-D,L-methionine (10) Through 3.3 ppm Se (ww) in eggs, decreased growth, Smith et a!., 1988 !;:, ;:,:

heron, embryo hatching increased hepatic TBARS concentration **~ ~** 

Mallard, duckling Sodium selenite (10) Day-old 5.0 ppm Se (ww) in liver, increased hepatic GSSG Heinz eta!., 1988; Hoffman .., !:!

through 6 weeks reductase activity and GSH concentration et a!., 1989 E'.

(20) 3.2 ppm Se (ww) in liver, decreased growth, 1)• **~**

increased hepatic GSH and GSSG concentrations,

"

decreased hepatic protein concentration 1)• 0

(40) 2.8 ppm Se (ww) in liver, decreased survival and C:l

growth,

increased plasma GSSG reductase activity,

**~** ...,

hepatic GSH, GSSG and TBARS concentrations, 'I

decreased

hepatic GSH peroxidase activity, hepatic t0 <:::>

total thiol, and protein concentrations §

Mallard, duckling Seleno-D,L-methionine (10) Day-old 4.8 ppm Se (ww) in liver, increased plasma and Heinz eta!., 1988; Hoffman ._ ......

through 6 weeks hepatic GSH peroxidase activities et a!., 1989 I t-v

(20) 26 ppm Se (ww) in liver, decreased growth, 0\

increased

plasma GSH peroxidase activity, hepatic

GSSG:GSH ratio, and TBARS concentration,

decreased

hepatic GSH, total thiol, and protein

concentrations

(40) 68 ppm Se (ww) in liver, decreased survival and

growth,

increased plamna GSSG reductase activity,

hepatic GSSG, GSSG:GSH ratio, TBARS concentration,

decreased hepatic GSH, total thiol

and

protein concentrations

Mallard, duckling Seleno-D,L-methionine (15) 4 weeks 10.8, 11.6, 14.9 ppm Se (ww) in liver, decreased Hoffman et al., 1991b,

growth, increased hepatic and plasma GSH 1992a,b

peroxidase activitiesTable I (Continued) Species, age Mallard, adult Mallard, adult Form of dietary Se (concentration, Observation

ppm) period

(60) Sodium selenite in drinking water 12 weeks

(3.5 mg/1) Selenomethionine in drinking water 12 weeks

(2.2 mg/1) Seleno-D,L-methionine (2) 14 weeks

(4) (8) (16) (32) Effects 49, 56, 72 ppm Se (ww) in liver, decreased survival

and growth, increased histopathological lesions,

plasma and hepatic GSH peroxidase activities,
GSSG:GSH ratio, TBARS, and plasma AST,

decreased plasma alkaline phosphatase activity,

hepatic GSH and total thiol concentrations References

Increased serum AL T Fairbrother and Fowles, 1990

5 ppm (ww) Se in liver, impaired immune function,

increased serum AL T, and plasma GSH peroxidase

activities

2 ppm Se (ww) in liver, increased plasma GSH Hoffman et al., 199la

peroxidase activity

4 ppm Se (ww) in liver, increased plasma GSH

peroxidase activity

12 ppm Se (ww) in liver, increased plasma GSH

peroxidase activity, hepatic GSSG concentration and

GSSG:GSH ratio

21 ppm Se (ww) in liver, decreased hemoglobin concentration, increased plasma GSH peroxidase

activity, hepatic GSH and TBARS concentrations

29 ppm Se (ww) in liver, decreased survival, body

weight, hematocrit, and hemoglobin concentration,

increased hepatic lesions, plasma GSH peroxidase and

alkaline phosphatase activities, total protein concentrations, hepatic GSH and TBARS concentrations, decreased DNA:RNA ratio b **~ ~**  §; § "--- "" **~** R· **~** " g· O' z;; **~** w 0 § ...... ......
I **~ ~**  Vl16 D.J. Hoffman/ Aquatic Toxicology 57(2002) 11-26



### fed the same amount ofSe. Results ofthis study suggested thatherons maybe less sensitive to the toxic effects ofSe than mallards.

2.2. Survival andgrowth in mallardducklings

Se as selenomethionine resulted in generally greater oxidative stress than did Se as selenite, as reflected by effects on hepatic glutathione metabolism and lipid peroxidation in mallard ducklings (Hoffman et al., 1989). These findings may reflect the much greater accumulation ofSe in the liver and other organs as dietary selenomethionine than as selenite. However, bothforms of Se reduced duckling growth and survival (Heinz et al., 1988). When day-old ducklings were fed 0.1, 10, 20, or40 ppmSeasseleno-D,L-methionine

or sodium selenite for 6weeks, Sefrom selenomethionineaccumulatedin adose-dependentmanner in the liver. Hepatic andplasma GSH peroxidase activity was initially elevated at 10 ppm Se as selenomethionine, whereas, GSSG reductase activity was elevated at higher dietary concentrations of Se. A decrease in the concentration of hepatic-reduced glutathione (GSH) and totalhepatic thiols (SH) at 20 ppm Se in the diet was accompanied by an increase in the ratio of oxidized glutathione (GSSG) to GSH, and an increase in TBARS concentration as evidence of lipid peroxidation (Hoffman et al., 1989). Se from selenite.accumulated in the liver to an apparent maximum at 10 ppmin the diet, resulting in an increase in hepatic GSH and GSSG, accompanied by a small decrease in hepatic total SH. Sodium selenite resulted in increased hepatic GSSGreductase activity at 10 ppmandinplasma GSSG reductase activity at 40 ppm. A small increase in lipid peroxidation occurred at 40 ppm. Further studies with Se in ducklings confirmed the previous findings for selenomethionine, includingdose-dependent oxidative stress (Hoffman et al., 199lb, 1992a,b). In these studies, decreased growth with increased plasma and hepatic GSH peroxidase activities occurred at 15 ppm Se in the diet. At 60 ppm Se in the diet, decreased survival and growth with increased histopathological lesions occurred. Other effects included, decreased plasma alkaline phosphatase act1v1ty related to decreased bone growth, and increased plasma aspartate aminotransferase (AST) activity related to hepatotoxicity. Pronounced oxidative stress included decreased hepatic GSH and total thiol concentrations with increased ratio of GSSG:GSH and TBARS concentration. Recent studieshave suggestedthatseleno-L-methionine may be more toxic to ducklings than seleno-D,L-methionine under certain dietary conditions; effects on survival and growth revealed that Se as seleno-L-methionine in a wheat-based diet resulted in higher mortality at 30 ppm Se than did either seleno-D,L-methionine or other Se groupsthatwere studied,includingselenizedyeast and wheat (Heinz et al., 1996; Hoffman et al., 1996). In contrast, when a different and commercially-based basal diet was provided, survival of ducklings was not affected by 30 ppm Se. Many of the previous avian toxicity studies with selenomethionine have used the mixture of stereoisomers, seleno-D,L-methionine, that was commercially available. Therefore, it is possible that under certain dietary conditions, the potential toxicity ofSe as naturally occurring seleno-Lmethionine may have been underestimated. Se as selenized yeast was found to be less toxic than seleno-D,L-methionine, seleno-L-methionine, or selenized wheat, as reflected by fewer effects on growth, plasma alkalinephosphataseactivity, and hepatic oxidative stress. Here, greater oxidative stressfor DL andLdietswasmanifestedby significantdecreases in hepatic GSH and total SH. This may berelated in part to the nearly double tissue accumulation of Se from selenomethionine diets than from selenized yeast.

2.3. Subchronic effects on immunefunction and hepatotoxicity in adult mallards

The subchroniceffects ofselenomethionine and sodium selenite on several immunologic, hematologic, and serologicparametersin adultmalemallards were measured, using concentrations in drinking water of0, 0.5, or 3.5 mgjl Se (Se) as sodium selenite; or 2.2 mg/1 (Se) as selenomethionine for 12 weeks (Fairbrother and Fowles, 1990). A battery ofin vivo and in vitro immuno-D.J. Hoffman/ Aquatic Toxicology 57(2002) 11-26 17
logic assays was performed oneachbirdthroughout the study. The selenomethionine-treatedbirds displayed animpaireddelayed-typehypersensitive (DTH)response to tuberculin (M. bovis), as measured by the number ofpositive reactions present 24 h post purified protein derivative (PPD) challenge. This group also exhibited increased serum alanine aminotransferase (ALT) andplasma GSH peroxidase activities. Se concentrationin the liver and breast muscle was significantly elevated 4- and 14-fold, respectively, over controls. Sodium selenite-treated birds did not display any detectable differences in immune function or Se accumulation in tissues above that of controls. SerumALT activity was increased in the 3.5 mg/1 group, although to a lesser extent than in selenomethionine-treated birds. Concentrations ofSe as sodium selenite didnot affect the immune system, whereas, low concentrations of selenomethionine (2.2 mgjl Se) appeared to suppress certain aspects ofthe mallard immune response. When adult male mallards received a control diet (0.2ppmSe) ordiets containing 1, 2, 4, 8, 16,

or 32 ppm Se as selenomethionine for 14 weeks, Se accumulated readily in the liver in a dose-dependent manner, reaching a mean concentration of29 ppm(ww) inthe 32 ppmgroup (Hoffmanet al., 199la). Dietary Se of 2 ppm or greater increased plasma glutathione peroxidase activity. Mortality (10%) and histopathological effects, including bile duct hyperplasia and hemosiderin pigmentation ofthe liver and spleen, occurred in the 32 ppmgroup. Thesehistopathological effects

were accompanied by lower hemoglobin concentrations (16 and 32 ppm groups) and hematocrit (32 ppm group), and elevated plasma alkaline phosphataseactivity (32 ppmgroup), indicativeof cholestatic liver injury. Manifestations ofhepatic oxidativestress included significantlineardose-responseincreasesin GSSGconcentrationandratio of GSSG:GSH with increasing Se; both ofthese measurements were significantly greater thancontrols for groups receiving 8-32 ppm Se. Mean hepatic GSH and TBARS concentrations were significantly elevated in the 16 and 32 ppm groups. 3. Field studies and selenium-related oxidative stress in aquatic birds

3.1. Effects on growth and immunefunction in avocet andstilt chicks

Excess Se in the aquatic food chain is embryotoxicandteratogenicto avocets, stilts, and other waterbirds. Normal growth andimmune function may also be compromised (Table 2). Avocets (Recurvirostra americana) hatched from eggs collectedfromthe southCentralValley ofCalifornia

were studied to determine the impact ofelevated concentrations of Se, ,arsenic, and boron (B) on the immune system and growthtomaturity (Fairbrother et al., 1994). The reference site for this study had low Se (1.2 ppb), As (29 ppb) and (B not measured) in the

**\.Vater~** The study site with the highest concentrations of Se ( 450 ppb ), also hadelevated As (127 ppb) and B (109 ppm). Size at hatch and growth rate of chicks was significantlyreduced atthehighest Se site. Chicks from thatsite exposed solely throughin ovo transfer of these elements had significantly increased heterophil:lymphocyteratios. Thephagocyticactivity ofmacrophages was also significantly reduced. In another study, hatchling morphology and oxidative stress were examined in american avocets and black-necked stilts (Himantopus mexicanus). Eggs were collectedfrom three sites in the Tulare Lake Basin of California and hatched in the laboratory (Hoffman et al., 2002). These sites included the Tulare LakeDrainageDistrict-north (TLDD-N, water 2.5 ppb Se), TLDD-south (TLDD-S, water 8.6 ppb Se) and Westfarmers (WF, water 190 ppb Se). Highest egg Se concentrations occurred atWF (geometricmean 9ppm,
wwfor avocets and 6ppmfor stilts), meanegg Se concentrationswere2ppmfor avocets and2.5for stilts atTLDD-S, and 1ppm for avocets and 0.7 for stilts atTLDD-N. Hatching success, incidence of malformations, body and organ weights and bone lengths did not differ significantly among locations. However, ratio ofliver to body weight

was greaterfor avocets from WF than TLDD-N. With increasing Se concentration, hepatic oxidative stress was most apparentin avocet hatchlings from WF in which GSH peroxidase activity in-**~** 00

Table 2 Field studies related to excess selenium and oxidative stress in aquatic birds

Species, age Location Tissue Se Effects References

(concentration, ppm)

American avocet (R. americana), hatchling Evaporation ponds in the 4.8 ppm (ww) in Malformations, decreased size at hatch, Fairbrother et al.,

through

adult Tulare Basin, CA eggs possible impaired immune function 1994 b

American avocet (R. americana), embryo, Evaporation ponds in the 9 [4-17] ppm (ww) Embryo growth, increased liver:body Hoffman et al.,

**~**

hatchling Tulare Basin, CA in eggs weight ratio, increased oxidative stress 1997 **~**

(increased hepatic GSH peroxidase, ratio §

GSSG:GSH, decreased G6PDH activity) **~** §

Black-necked stilt (H. mexicanus), Evaporation ponds in the 6 [1.5-15] ppm Decreased hepatic GSH Hoffman et al., "'-._

embryo,

hatchling Tulare Basin, CA (ww) in eggs 1997 ::t:.. "" :::

America coot (Fulica americana), adult Kesterson Reservoir, CA. 28 ppm (ww) in Emaciation, abnormal feather loss, Ohlendorf et al., ;;

liver

histopathological lesions, decreased hepatic 1988 p;·

glycogen and protein, increased oxidative

**~ ~** 

stress

(hepatic GSH peroxidase, total thiol, p;· <:>

PBSH,

GSH)cSurf

scoter (Melanitta perspicillata), adult San Francisco Bay, CA 36 [17-60] ppm Positive correlation between hepatic GSH Hoffman et a!., l'.;l v.

(ww) in liver peroxidase and hepatic Se concentration 1998 'I

(r = 0.63, P<0.05) w <::::>

Ruddy duck ( Oxyura jamaicensis ), adult San Francisco Bay, CA 12 [2-21] ppm Decreased hepatic GSH with increased Se Hoffman et al., ,§

(ww) in liver (r = -0.740, P<0.05) 1998

.....

Emperor

geese (C. canagica), adult Alaska 2.9 [0.1-9.4] ppm Positive correlation between plasma GSH Hoffman, .....
I **~**

(ww) in blood peroxidase and blood Se concentration unpublished data 0\

(r = 0.62, P<O.OOl), negative correlation

between plasma GSSG reductase and

blood Se concentration (r = -0.44,

P<0.01)

Willet (C. semipalmatus ), adult San Diego, CA 1.3 [0.2--4.5] ppm Positive correlation between hepatic Se Hoffman and Hui,

(ww)

in liver concentration and the following: GSSG unpublished data

(r=0.70,

P<0.001), GSSG:GSH (r=0.57,

P<0.01), and TBARS (r = 0.55, P<0.05)D.J. Hoffman/ Aquatic Toxicology 57(2002) 11-26 19



### creased, glucose-6-phosphate dehydrogenase (G6-PDH) decreased, and GSSG concentration increased, as well as, the ratio of GSSG to GSH. In stilts, hepatic GSH was lower in WF hatchlings, with few other effects.

3.2. Selenium toxicosis in adult american coots



### Severe gross and microscopic lesions and other changes found in american coot embryos and adults from Kesterson Reservoir, Merced County, CA were attributed to excess Se, readily identified in the water, food items, and tissues ofthe birds (Ohlendorfet al., 1988). Adult birds

were emaciated, had subacute to extensive chronic hepatic lesions and excess fluid and fibrin in the peritoneal cavity. Biochemical changes in their livers included elevated glutathione peroxidase activity, glycogen and GSH concentrations, but lower protein, total sulfhydryl, and protein-bound sulfhydryl concentrations. Congenital malformations observed grossly in 

### embryos were often multiple and included anophthalmia, micropthalmia, abnormal beaks, amelia, micromelia, ectrodactyly, and hydrocephaly. Mean concentrations of Se in livers (28 ppm, ww) and kidneys (29 ppm) of adult coots collected at the Kesterson ponds were about 10 times those found at a nearby control

area (2.5 and 3.7ppm).

3.3. Selenium andglutathione metabolism in diving ducks



### Adult male greater scaup (Aythya marila) 

### (GS), surf scoters (Melanitta perspicillata) 

### (SS), and ruddy ducks (Oxyura jamaicensis) 

### (RD)

were collected from Suisun Bay and coastal Tomales Bay in the greater San Francisco Bay

area to assess exposure to inorganic contaminants. Hepatic Se concentrations were highest in GS (geometric mean=67 ppm, dw) and SS (119 ppm) in Suisun Bay, whereas, hepatic mercury (Hg) was highest (19 ppm) in GS and SS from Tomales Bay. Hepatic Se and Hg were lower in RD and did not differ between locations. Hepatic GSH-peroxidase activity was higher in SS and RD, and G-6-PDH higher in GS and SS from Suisun Bay than Tomales Bay. GSSG-reductase was higher in SS from Suisun Bay. The ratio of GSSG:GSH was greater in all species from Tomales Bay. The following significant relationships were found in one or more species with increasing hepatic Hg concentration: body, liver and heart weights, hepatic GSH concentration, G-6-PDH and GSH-peroxidase activities decreased whereas the ratio of GSSG to GSH, and GSSG-reductase increased. With increasing hepatic Se concentration, GSH-peroxidase mcreased but GSH decreased.

3.4. Elevated bloodselenium andplasma enzymes ofemperor geese



### As high as 10 ppm Se (ww) was found in blood samples collected from incubating female 

### emperor geese (Chen canagica) 

### on their breeding grounds of the Yukon-Kuskokwim Delta in western Alaska (Franson et al., 1999). Incubating adult females captured in late May through mid-June 1997 had higher concentrations of Se in their blood (mean=5.60 ppm) than adult females captured during wing molt in late-July 1996 (mean=2.78 ppm). Females that nested early or were in good body condition had higher concentrations of Se in their blood than did other nesting females. Blood samples from 4 of 29 goslings had detectable levels of Se (mean=0.14 ppm). Findings suggest that emperor geese are exposed to more Se in the marine environment of their wintering and staging areas on the Alaska Peninsula than

on the breeding grounds. Mercury levels were generally low since the highest concentration of Hg found in the blood of emperor geese was 0.24 ppm in an adult female during wing molt, but the mean concentration of Hg was 0.05 ppm. Hg was not correlated with Se. During the subsequent year, as high as, 11.3 ppm Se was found in the blood of incubating females (mean=5.0 ppm) and a positive correlation between blood Se concentration and plasma GSH peroxidase activity was found but

a negative correlation was observed with plasma GSSG reductase activity (Hoffman, unpublished data).20 D.J. Hoffman/ Aquatic Toxicology 57(2002) 11-26

3.5. Selenium andglutathione metabolism in willets



### Willets (Catoptrophorus semipalmatus) 

### that

were overwintering in southern California near SanDiego were found throughanalysis ofingesta to be feeding primarily onmollusks andcrustacea (Hui andBeyer, 1998). Liver Se concentrations of the willets were found to containup to 4.5 pm Se (ww), and positive correlations were found between hepatic Se concentration and ratio ofhepatic GSSG:GSH, and and TBARS concentration (Hoffman and Hui, unpublished data).

4. Discussion



### Antioxidants are a major resource ofmost living organisms for protection against diverse free radical and other oxidative stressors, including

excess selenium, to which they invariably become exposed (Cross et al., 1987; Griffith, 1999). Glutathioneis an importantantioxidant andcofactor for antioxidant enzymes; high electron-donating capacity combinedwithhighintracellular concentration endows GSH with great reducing power, which is used to regulate a complex thiol-exchange system

**(-SH~-s-S-).** 

### This functions at all levels ofcell activity, from therelatively simple (circulating cysteinej-SH thiols, ascorbate, other small molecules) to the most complex (cellular -SH proteins) (Kidd, 1997). Changes in GSH status including depletion are thought to constitute an important component of injury to liver and other tissues. As cellular GSH is depleted, individual cells first die in local areas most affected, followed by zones oftissue damage often occurringwhere contentofpolyunsaturatedlipids

are highest and/or antioxidant defenses are most meager. Therefore, repletion of glutathione appears to be central to intrinsic adaptive strategies for meeting the challenge ofsustained (or acute) oxidative stress (Levine and Kidd, 1985; Kidd, 1993).

In keeping with the above, many ofthe avian studies described within this review havereported hepatic oxidative stress at different stages ofthe life cycle due to excess dietary Se, mainly in the organic form (seleno-D,L-methionine, seleno-Lmethionine or Se incorporated into wheat or yeast). Manifestations ofthe oxidative stress included alterations in hepatic glutathione metabolism towards a more oxidized state, accompanied by lipid peroxidation. As dietary and subsequent tissue concentrations ofSe increased, plasma and hepatic GSHperoxidase activities increased, followed by dose-dependent increases in the ratio of hepatic oxidized to reduced glutathione (GSSG:GSH), and ultimately increased hepatic lipid peroxidation. These dose-related effects were apparent in the four studies with mallard ducklings (Hoffman et al., 1989, 1992a,b, 1996) and two studies with adult mallards (Fairbrotherand Fowles, 1990; Hoffmanet al., 1991a). Elevated plasma GSH-peroxidase activity is an indicator ofSe exposure and the onset ofoxida

### tive stress inbirds (Hoffman et al., 1989, 1991a,b) and mammals (Combs and Combs, 1986; Whanger, 1986; Levander, 1986). Inmallard studies, two factors accounted for the dose-dependent increase inratio ofGSSGto GSH: (1) 

### a dose-dependent increase of oxidized glutathione by, as much as, 240%; and (2) a dose-dependent depletion ofreduced glutathione(GSH)withincreasing concentrations ofhepatic Se, sometimes falling to less than 25% ofcorresponding control values. A depletion oftotal hepaticthio1s often appeared to parallel the depletion of GSH but was, as much
as, twice that ofthe GSH depletion in terms of micromoles ofGSH equivalents. Related hepatic lesions occurred andincluded hepaticcell necrosis and bile duct hyperplasia. Similar findings have been reported for Se as seleniteunderinvitro conditionswith isolatedrat hepatocytes where partial to total depletion of thiols occurred with a concomitant increase in GSSG and 0 2 

### consumption (Anundiet al., 1984). Excess GSSG can react with protein sulfhydryls, contributing to the total thiol and proteinbound thiol depletions, observed in mallard studies, by the formation of mixed glutathione: protein disulfides. Formation ofmixed disulfi.des may be part of a significant mechanism in regulating metabolic activity as well as integrityofcellmembranes in response to oxidative stress (Keeling et al., 1982; Ondarza, 1989; Kidd, 1997). InmallardD.J. Hoffman/Aquatic Toxicology 57(2002) 11-26 21


### duckling studies, excess dietary Se as selenomethionine had a more pronounced effect than sodium selenite on hepatic glutathione metabolism and lipid peroxidation as reflected by greaterGSH and total SH depletions. Differences in responsebetweenthe two forms mayberelated in part to the much higher hepatic accumulation ofSe as selenomethionine.Whenexcess dietary Se

was provided as sodium selenite to ducklings, an increase in hepatic GSH occurred, accompanied by anincreasein GSSGsimilartothatreportedin laboratory rodents (LeBoeuf et al., 1985). However, the GSSGto GSHratio didnotsignificantly increase, presumably due to an adaptive increase in GSH, whichmostlikelyrepresented an attempt to maintain a nearly normal ratio of GSSG to GSH (LeBoeuf and Hoekstra, 1983). Elevated GSSGreductase activity was consistent with this observation. Furthermore, selenite had less of a stimulatory effect than selenomethionine on lipid peroxidationin ducklings, which would appearto be a further reflection ofthe adaptive increase in GSH. Such an increase in reducing equivalents would combat lipid peroxidation, in contrast to the large depletion ofGSH and total SH caused by selenomethionine. A similar hepatic Se accumulationpatternfor selenitehas beenreportedin young chickens in which a maximum accumulation occurred at 10 ppm dietary Se with a slight decrease at higher dietary levels (Jensen, 1975). Manifestations of Se-related oxidative effects

on glutathione metabolism were also apparent in field studies in seven species of aquatic birds at different stages ofthe life cycle including embryonic development. Exposure during embryonic development to a variety of environmental contaminants is known to cause in ovo and in utero deathofembryos andfetuses, andto ofteninitiate birth defects (teratogenesis) in the surviving offspring. Yet, limited understanding exists on the exact biochemical mechanisms, or the determinants of teratological susceptibility. Recent in vitro and in vivo studies primarily based on rodent models suggest that many potential embryotoxicxenobiotics are actually proteratogensthat must be bioactivated by enzymes including cytochromes P450 or by peroxidases such as prostaglandin H synthase to produce reactive intermediarymetabolitesthat are teratogenic (Wells and Winn, 1996). These reactive intermediates generally are electrophiles or free radicals that bind covalently (irreversibly) to, and either directly or indirectly oxidize embryonic cellular macromolecules, such as, DNA, protein, and lipid, irreversibly alteringcellularfunction. Target oxidation, known as oxidative stress, often appears to be mediated by reactive oxygen species (ROS), such as, hydroxyl radicals. Largely due to immature or otherwise compromised embryonic pathways for detoxification, cytoprotection and repair, theembryois relatively susceptibleto reactive intermediates, and teratogenesis via this mechanism can occureven from exposureto therapeutic concentrations of drugs or supposedly safe concentrations of environmental chemicals, including Se. In keeping with the above, studies withculturedratembryos thatexaminetheeffects of depletion of reduced glutathione (GSH) on selenium (Se) embryotoxicity concluded that embryonic GSH status plays a role in the embryotoxicity of selenite and selenate (Usami et al., 1999). There are several mechanisms in which selenium may express its embryotoxicity and teratogenicity to the avian embryo. One possible mechanism would involve tissue injury as a result ofoxidative stress, possibly with theformation of CH3Se which either enters a redox cycle and generates superoxide and oxidative stress, or forms free radicals thatbindto andinhibitimportant enzymes and proteins (Spallholz and Hoffman, 2002). Largelyduetoimmatureorotherwise compromised embryonic pathways for detoxification, cytoprotection and repair, the embryo is relatively susceptible to reactive intermediates and teratogenesis via this mechanism. In order to test this hypothesis further, feeding studies or egg injection studies could be done with Se as selenomethionine under conditions ofrestricted antioxidants or possibly containing an inhibitor of GSH synthesis such as buthionine sulfoximine (BSO). Comparisons could be conducted with baseline controls, as well as othergroups containing highly supplemented antioxidants including glutathione and/or its precursors, as well as vitamins, such as C and E. Another possible mechanism for selenium teratogenesis in birds may be22 D.J. Hoffman/Aquatic Toxicology 57(2002) 11-26



### related to the presence ofexcess selenium analogs of sulfur-containing enzymes and structural proteins as a consequence of high levels of selenomethionine in the diet. This hypothesis could perhaps befurther testedbyexaminingthe effects of other methionine analogues on development for potential teratogenesis includingtype and frequency ofmalformation and for percent impairment of hatching success by means of egg injection and ultimately feeding studies with mallards. In addition to hepatotoxicity and teratogenesis other potentially toxic and biologically relevant effects of excess selenium have been reported in avian laboratory and field studies. One ofthese effects includes reduced immune function which occurred in conjunction with oxidative stress (Fairbrother andFowles, 1990; Fairbrotheret al., 1994) In mammals, the proliferation, growth and differentiation of immune cells is dependent on GSH and is most likely so in birds as well. Both T and B lymphocytes require adequate levels of intracellular GSH to differentiate, and with relatively low lymphocyte GSH concentrations were found to have significantly lower CD4 counts (Kinscherfet al., 1994). Intracellular GSH is also required for the T-cell proliferative response to mitogenic stimulation, for the activation ofcytotoxic T 'killer' cells, (Droge et al., 1994) and for many specific T-cell functions, including DNA synthesis for cell replication, as well as for the metabolism of interleukin-2 which is important for the mitogenic response (Wu et al., 1994). Experimental depletion ofGSH inhibits immune cell functions, sometimes markedly and in a number ofdifferent experimental systems the intracellular GSH of lymphocytes was shown to determinethemagnitudeofimmunologicalcapacity (Fidelus and Tsan, 1987; Droge et al., 1994). These and other findings indicate that intracellular GSH status plays a central role in the functioning ofimmune cells, and that disturbances in GSH status by selenium could be potentially disruptive. Furtherstudies areneeded to examinethe relationship between different forms of environmentally occurring selenium as well as interaction withotherelementsincludingarsenic andmercury
on immune function. When evaluating different aspects of Se toxicity, including biological effects and oxidative stress, interactive nutritional factors appear to play an important role. Given the great seasonal and species variability in diets ofwild birds, including aquatic birds, there is good reason to believe that different forms of Se may interact with nutritional variables in thefield to affect the health ofbirds. Important interactive nutritional factors studiedin mallards haveincluded: concentrations of dietary protein, methionine, and certain other elements, such as As, B and Hg (Hoffman et al., 199lb, 1992a,b; Heinz, 1996; Heinz and Hoffman, 1998; Stanley et al., 1996). Adequate dietary protein, supplemental methionine,As andHgappearto act antagonisticallywith Se in mallards, ameliorating adverse effects including: mortality, impaired gro\vth, histopatho

### logical lesions, and manifestations of oxidative stress. Arsenate alone reduces hatching success and hatchling survival, but in combination with Se ameliorates Se-relatedreproductiveimpairment and teratogenesis (Stanley et al., 1994). When the interaction ofarsenic and selenium at the subcellular level in rat kidney cells was studied using electronicprobemicroanalysis thatpermits detection of elements in the intracellular organelles, arsenic and selenium were concentrated and precipitated inthe lysosomes ofthe renal cells in the form of insoluble selenide (As2Se) (Berry and Galle, 1994). In thelong term, the lysosomes and their precipitate are eliminated in the urine. This intralysosomal concentration and precipitation may serve as a model for the mechanism of interaction of selenium in other tissues and species. Diving ducks in the San Francisco Bay area exhibited positive correlationsbetween hepatic Se concentrationandGSHperoxidase activity, buta negative correlationbetweenhepatic Se and GSH concentration (Hoffman et al., 1998). However, high concentrations of Hg were a confounding factor in this study. Interestingly, in willets in the San Diego area where little Hg was apparent, a positivecorrelationbetweenhepatic Seconcentration and oxidative stress occurredwith onlymodestly elevated hepatic Se concentrations (Hui et al., unpublished data). Similarlyin emperorgeese, there was little apparent blood Hg present, butD.J. Hoffman/ Aquatic Toxicology 57(2002) 11-26 23

elevated blood Se concentration, resulting in elevated plasma GSH peroxidase activity and decreased plasma GSSG reductase (Hoffman, unpublished data). In adult mallards combinations of methylmercury and selenomethionine

were antagonistic, but appeared additive or synergistic in toxcity to mallard embryos in terms of embryo survival and teratogenisis (Hoffman and Heinz, 1998). In mammalian studies, when selenium compounds were coadministered with mercuric mercury, a remarkable depression was noted in the acute toxicity of mercury through reduction of mercury accumulation in the kidneys, prolonged retention of mercury and selenium as inert high molecular weight complexes in the blood, particularly in the erythrocytes, and the formation of stable and nondiffusible complexes of mercury and selenium in various organs such as the liver and kidneys (Imura and Naganuma, 1991). Further investigation in birds is warranted to compare the interactions ofvarious forms of mercury and selenium particularly with respect to effects on reproduction. Other selenium and nutritional interaction studies are called for that would optimize GSH and other antioxidant defenses in a manner that would stabilize or raise the cell's threshold for susceptibility to toxic attack from excess selenium. Possible dietary means for attaining this include increasing dietary concentration of one or more of the following: GSH, L-methionine, 'activated' methionine metabolite known as SAM (Sadenosyl methionine), L-cysteine, N-acetyl cysteine (NAC), as well as glycine, L-glutamine, and L-taurine. Optimizing antioxidant capacity through other sources including vitamins E and C could also prove to be beneficial.

5. Conclusion
Laboratory studies, mainly with Se as organic selenomethionine, have revealed oxidative stress in different stages of the mallard (Anas platyrhynchos) life cycle. As dietary and tissue concentrations of Se increase, increases in plasma and hepatic GSH peroxidase activities
occur, followed by dose-dependent increases in the ratio of hepatic GSSG:GSH, and ultimately hepatic lipid peroxidation. One or more ofthese oxidative effects were associated with teratogenesis (4.6 ppm wet weight Se in eggs), reduced growth in ducklings (15 ppm Se in liver), diminished immune function in adults (5 ppm Se in liver) and histopathological lesions (29 ppm Se in liver). Manifestations of Se-related effects on glutathione metabolism have also been documented in field studies in seven species of aquatic birds, including american avocets, black-necked stilts, american coots, several species of diving ducks, emperor geese and willets. Accompanying biological effects in these species ranged from reduced growth and immune function to emaciation, histopathological lesions, and mor

### tality. There are several mechanisms in 

### vlhich selenium may express its embryotoxicity and teratogenicity to the avian embryo that require further investigation, including: (1) tissue injury as
a result ofoxidative stress; and (2) the action of selenium analogs of sulfur-containing enzymes and structural proteins as a consequence ofhigh levels of selenomethionine in the diet. Intracellular GSH status plays a central role in the functioning of immune cells and disturbances in GSH status by selenium could be potentially disruptive. Therefore, fur!Jler studies are

**ne~** to examine the

**relations~rent** 

### £- s o environmentally occurring selenium as well as interaction with other elements incl

rsemc an mercury on immune function. Further investigation in birds is warranted to compare the interactions of various forms of mercury and selenium particularly with respect to effects on reproduction. Other selenium and nutritional interaction studies are called for that would optimize GSH and other antioxidant defenses in a manner that would stabilize or raise the cell's threshold for susceptibility to toxic attack from excess selenium. In conclusion, Se-related manifestations ofoxidative stress may provide an insight into the nature of different aspects of selenium related toxicosis in birds and serve as useful bioindicators of Se exposure and toxicity in wild aquatic birds.24 D.J. Hoffman/ Aquatic Toxicology 57(2002) 11-26
Acknowledgements



### E.C. Colket and C.A. Erwin provided valuable assistance in the preparation ofthis review.

References

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